Antisense-oligonucleotides as inhibitors of tgf-r signaling

ABSTRACT

The present invention relates to antisense-oligonucleotides having a length of at least 10 nucleotides, wherein at least two of the nucleotides are LNAs, their use as inhibitors of TGF-R signaling, pharmaceutical compositions containing such antisense-oligonucleotides and the use for prophylaxis and treatment of neurological, neurodegenerative, fibrotic and hyperproliferative diseases.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

Any and all applications for which a foreign or domestic priority claimis identified in the Application Data Sheet as filed with the presentapplication are hereby incorporated by reference under 37 CFR 1.57.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to antisense-oligonucleotides, their useas inhibitors of TGF-R signaling, pharmaceutical compositions containingsuch antisense-oligonucleotides and the use for prophylaxis andtreatment of neurological, neurodegenerative and hyperproliferativeincluding oncological diseases.

Description of the Related Art

TGF-β exists in three known subtypes in humans, TGF-β1, TGF-β2, andTGF-β3. These are upregulated in neurodegenerative diseases, such asALS, and some human cancers, and increased expression of this growthfactor in pathological conditions of neurodegenerative diseases, acutetrauma, and neuro-inflammation and ageing has been demonstrated.Isoforms of transforming growth factor-beta (TGF-β1) are also thought tobe involved in the pathogenesis of pre-eclampsia.

Activated TGF-βs exert their effects on the target cell via threedifferent receptor classes: type I (TGFRI), also termed activin-likekinases (ALK; 53 kDa), type II (TGFRII; 70-100 kDa), and type III(TGFRIII; 200-400 kDa. TGF-β receptors are single pass serine/threoninekinase receptors. Whereas type II receptor kinase is constitutivelyactive, type I receptor needs to be activated. This process is initiatedthrough binding of a ligand to TGFRII; this triggers the transientformation of a complex that includes the ligand and receptor types I andII. Taking into account the dimeric composition of the ligand, thereceptor complex most likely consists of a tetrameric structure formedby two pairs of each receptor type.

TGF-β signal transduction takes place through its receptors anddownstream through Smad proteins. Smad-dependent cellular signaltransduction initiated by binding of the TGF-β isoform to a specificTGFRI/II receptor pair, leads to the phosphorylation of intracellularSmads and subsequently the translocation of an activated Smad complexinto the nucleus in order to influence specific target gene expression.Signal divergence into other pathways and convergence from neighboringsignaling pathways generate a highly complex network. Depending on theenvironmental and cellular context, TGF-beta signaling results in avariety of different cellular responses such as cellular proliferation,differentiation, motility, and apoptosis in tumor cells. In cancer,TGF-β can affect tumor growth directly (referred to as intrinsic effectof TGF-β signaling) or indirectly (referred to as extrinsic effect) bypromoting tumor growth, inducing epithelial-mesenchymal transition(EMT), blocking antitumor immune responses, increasing tumor-associatedfibrosis, modulating extracellular matrix (ECN) and cell migration, andfinally enhancing angiogenesis. The factors (e.g. concentration, timing,local exposure) determining whether TGF-β signaling has a tumor promoteror suppressor function are a matter of intense research and discussion.Currently, it is postulated that the tumor suppressor function of TGF-βsignaling is lost in early stages of cancer similar to recessiveloss-of-function mutations in other tumor suppressors. Therefore thereare several pharmacological approaches for treatment of divers cancersby blocking TGF-beta signaling pathways, such as investigation ofGalunisertib and TEW-7197, both are small molecule inhibitor of TGFRIand being in clinical investigation, and LY3022859, an antibody againstTGFRII.

Signals provided by proteins of the transforming growth factor (TGF-β)family represent a system by which neural stem cells are controlledunder physiological conditions but in analogy to other cell types arereleased from this control after transformation to cancer stem cells.TGF-β is a multifunctional cytokine involved in various physiologicaland patho-physiological processes of the brain. It is induced in theadult brain after injury or hypoxia and during neurodegeneration when itmodulates and dampens inflammatory responses. After injury, althoughTGF-β is in general neuroprotective, it limits the self-repair of thebrain by inhibiting neural stem cell proliferation and inducingfibrosis/gliosis for scar formation. Similar to its effect on neuralstem cells, TGF-β reveals anti-proliferative control on most cell types;however, paradoxically, many tumors escape from TGF-β control. Moreover,these tumors develop mechanisms that change the anti-proliferativeinfluence of TGF-β into oncogenic cues, mainly by orchestrating amultitude of TGF-β-mediated effects upon matrix, migration and invasion,angiogenesis, and, most importantly, immune escape mechanisms. Thus,TGF-β is involved in tumor progression (see FIG. 3).

Consequently, the TGF Receptor II (transforming growth factor, betareceptor II; synonymously used symbols: TGF-beta type II receptor,TGFBR2; AAT3; FAA3; LDS1B; LDS2; LDS2B; MFS2; RIIC; TAAD2; TGFR-2;TGFbeta-RII, TGF-RII, TGF-R_(II)), and in particular its inhibition, wasvalidated as target for the treatment of neurodegenerative diseases,such as ALS, and hyperproliferative diseases such as cancer and fibroticdiseases.

SUMMARY OF THE INVENTION

Thus objective of the present application is to provide pharmaceuticallyactive compounds able inhibit expression of the TGF Receptor II(TGF-R_(II)) and therefore, reduce the amount of TGF Receptor II(TGF-R_(II)) and decrease the activity of TGF-6 downstream signaling.

The objective of the present invention is solved by the teaching of theindependent claims. Further advantageous features, aspects and detailsof the invention are evident from the dependent claims, the description,the figures, and the examples of the present application.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Surprisingly under thousands of candidate substances, such asprotein-nucleotide complexes, siRNA, microRNA (miRNA), ribozymes,aptamers, CpG-oligos, DNA-zymes, riboswitches, lipids, peptides, smallmolecules, modifyers of rafts or caveoli, modifyers of golgi apparatus,antibodies and their derivatives, especially chimeras, Fab-fragments,and Fc-fragments, antisense-oligonucleotides containing LNAs (LNA®:Locked Nucleic Acids) were found the most promising candidates for theuses disclosed herein.

Thus, the present invention is directed to antisense-oligonucleotide(s)consisting of 10 to 28 nucleotides and at least two of the 10 to 28nucleotides are LNAs and the antisense-oligonucleotide is capable ofhybridizing with a region of the gene encoding the TGF-R_(II) or with aregion of the mRNA encoding the TGF-R_(II), wherein the region of thegene encoding the TGF-R_(II) or the region of the mRNA encoding theTGF-R_(II) comprises the sequence TGGTCCATTC (Seq. ID No. 4) or thesequence CCCTAAACAC (Seq. ID No. 5) or the sequence ACTACCAAAT (Seq. IDNo. 6) or the sequence GGACGCGTAT (Seq. ID No. 7) or the sequenceGTCTATGACG (Seq. ID No. 8) or the sequence TTATTAATGC (Seq. ID No. 9),and the antisense-oligonucleotide comprises a sequence capable ofhybridizing with said sequence TGGTCCATTC (Seq. ID No. 4) or sequenceCCCTAAACAC (Seq. ID No. 5) or sequence ACTACCAAAT (Seq. ID No. 6) orsequence GGACGCGTAT (Seq. ID No. 7) or sequence GTCTATGACG (Seq. ID No.8) or sequence TTATTAATGC (Seq. ID No. 9) respectively and salts andoptical isomers of said antisense-oligonucleotide.

Slightly reworded the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of theopen reading frame of the gene encoding the TGF-R_(II) or with a regionof the mRNA encoding the TGF-R_(II), wherein the region of the openreading frame of the gene encoding the TGF-R_(II) or the region of themRNA encoding the TGF-R_(II) comprises the sequence TGGTCCATTC (Seq. IDNo. 4) or the sequence CCCTAAACAC (Seq. ID No. 5) or the sequenceACTACCAAAT (Seq. ID No. 6) or the sequence GGACGCGTAT (Seq. ID No. 7) orthe sequence GTCTATGACG (Seq. ID No. 8) or the sequence TTATTAATGC (Seq.ID No. 9), and the antisense-oligonucleotide comprises a sequencecapable of hybridizing with said sequence TGGTCCATTC (Seq. ID No. 4) orsequence CCCTAAACAC (Seq. ID No. 5) or sequence ACTACCAAAT (Seq. ID No.6) or sequence GGACGCGTAT (Seq. ID No. 7) or sequence GTCTATGACG (Seq.ID No. 8) or sequence TTATTAATGC (Seq. ID No. 9) respectively and saltsand optical isomers of said antisense-oligonucleotide.

Alternatively the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceTGGTCCATTC (Seq. ID No. 4) or the sequence CCCTAAACAC (Seq. ID No. 5) orthe sequence ACTACCAAAT (Seq. ID No. 6) or the sequence GGACGCGTAT (Seq.ID No. 7) or the sequence GTCTATGACG (Seq. ID No. 8) or the sequenceTTATTAATGC (Seq. ID No. 9), and the antisense-oligonucleotide comprisesa sequence complementary to the sequence TGGTCCATTC (Seq. ID No. 4) orthe sequence CCCTAAACAC (Seq. ID No. 5) or the sequence ACTACCAAAT (Seq.ID No. 6) or the sequence GGACGCGTAT (Seq. ID No. 7) or the sequenceGTCTATGACG (Seq. ID No. 8) or the sequence TTATTAATGC (Seq. ID No. 9)respectively and salts and optical isomers of saidantisense-oligonucleotide.

Slightly reworded the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of theopen reading frame of the gene encoding the TGF-R_(II) or with a regionof the mRNA encoding the TGF-R_(II), wherein the region of the openreading frame of the gene encoding the TGF-R_(II) or the region of themRNA encoding the TGF-R_(II) comprises the sequence TGGTCCATTC (Seq. IDNo. 4) or the sequence CCCTAAACAC (Seq. ID No. 5) or the sequenceACTACCAAAT (Seq. ID No. 6) or the sequence GGACGCGTAT (Seq. ID No. 7) orthe sequence GTCTATGACG (Seq. ID No. 8) or the sequence TTATTAATGC (Seq.ID No. 9), and the antisense-oligonucleotide comprises a sequencecomplementary to the sequence TGGTCCATTC (Seq. ID No. 4) or the sequenceCCCTAAACAC (Seq. ID No. 5) or the sequence ACTACCAAAT (Seq. ID No. 6) orthe sequence GGACGCGTAT (Seq. ID No. 7) or the sequence GTCTATGACG (Seq.ID No. 8) or the sequence TTATTAATGC (Seq. ID No. 9) respectively andsalts and optical isomers of said antisense-oligonucleotide.

Preferably the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceTGGTCCATTC (Seq. ID No. 4), and the antisense-oligonucleotide comprisesa sequence capable of hybridizing with said sequence TGGTCCATTC (Seq. IDNo. 4) and salts and optical isomers of said antisense-oligonucleotide.

Slightly reworded the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of theopen reading frame of the gene encoding the TGF-R_(II) or with a regionof the mRNA encoding the TGF-R_(II), wherein the region of the openreading frame of the gene encoding the TGF-R_(II) or the region of themRNA encoding the TGF-R_(II) comprises the sequence TGGTCCATTC (Seq. IDNo. 4), and the antisense-oligonucleotide comprises a sequence capableof hybridizing with said sequence TGGTCCATTC (Seq. ID No. 4) and saltsand optical isomers of said antisense-oligonucleotide.

Alternatively the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceTGGTCCATTC (Seq. ID No. 4), and the antisense-oligonucleotide comprisesa sequence complementary to the sequence TGGTCCATTC (Seq. ID No. 4) andsalts and optical isomers of said antisense-oligonucleotide.

Slightly reworded consisting of 10 to 28 nucleotides and at least two ofthe 10 to 28 nucleotides are LNAs and the antisense-oligonucleotide iscapable of hybridizing with a region of the open reading frame of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the open reading frame of the geneencoding the TGF-R_(II) or the region of the mRNA encoding theTGF-R_(II) comprises the sequence TGGTCCATTC (Seq. ID No. 4), and theantisense-oligonucleotide comprises a sequence complementary to thesequence TGGTCCATTC (Seq. ID No. 4) and salts and optical isomers ofsaid antisense-oligonucleotide.

Preferably the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceCCCTAAACAC (Seq. ID No. 5), and the antisense-oligonucleotide comprisesa sequence capable of hybridizing with said sequence CCCTAAACAC (Seq. IDNo. 5) and salts and optical isomers of said antisense-oligonucleotide.

Slightly reworded the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of theopen reading frame of the gene encoding the TGF-R_(II) or with a regionof the mRNA encoding the TGF-R_(II), wherein the region of the openreading frame of the gene encoding the TGF-R_(II) or the region of themRNA encoding the TGF-R_(II) comprises the sequence CCCTAAACAC (Seq. IDNo. 5), and the antisense-oligonucleotide comprises a sequence capableof hybridizing with said sequence CCCTAAACAC (Seq. ID No. 5) and saltsand optical isomers of said antisense-oligonucleotide.

Alternatively the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceCCCTAAACAC (Seq. ID No. 5), and the antisense-oligonucleotide comprisesa sequence complementary to the sequence CCCTAAACAC (Seq. ID No. 5) andsalts and optical isomers of said antisense-oligonucleotide.

Slightly reworded consisting of 10 to 28 nucleotides and at least two ofthe 10 to 28 nucleotides are LNAs and the antisense-oligonucleotide iscapable of hybridizing with a region of the open reading frame of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the open reading frame of the geneencoding the TGF-R_(II) or the region of the mRNA encoding theTGF-R_(II) comprises the sequence CCCTAAACAC (Seq. ID No. 5), and theantisense-oligonucleotide comprises a sequence complementary to thesequence CCCTAAACAC (Seq. ID No. 5) and salts and optical isomers ofsaid antisense-oligonucleotide.

Preferably the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceACTACCAAAT (Seq. ID No. 6), and the antisense-oligonucleotide comprisesa sequence capable of hybridizing with said sequence ACTACCAAAT (Seq. IDNo. 6) and salts and optical isomers of said antisense-oligonucleotide.

Slightly reworded the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of theopen reading frame of the gene encoding the TGF-R_(II) or with a regionof the mRNA encoding the TGF-R_(II), wherein the region of the openreading frame of the gene encoding the TGF-R_(II) or the region of themRNA encoding the TGF-R_(II) comprises the sequence ACTACCAAAT (Seq. IDNo. 6), and the antisense-oligonucleotide comprises a sequence capableof hybridizing with said sequence ACTACCAAAT (Seq. ID No. 6) and saltsand optical isomers of said antisense-oligonucleotide.

Alternatively the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceACTACCAAAT (Seq. ID No. 6), and the antisense-oligonucleotide comprisesa sequence complementary to the sequence ACTACCAAAT (Seq. ID No. 6) andsalts and optical isomers of said antisense-oligonucleotide.

Slightly reworded consisting of 10 to 28 nucleotides and at least two ofthe 10 to 28 nucleotides are LNAs and the antisense-oligonucleotide iscapable of hybridizing with a region of the open reading frame of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the open reading frame of the geneencoding the TGF-R_(II) or the region of the mRNA encoding theTGF-R_(II) comprises the sequence ACTACCAAAT (Seq. ID No. 6), and theantisense-oligonucleotide comprises a sequence complementary to thesequence ACTACCAAAT (Seq. ID No. 6) and salts and optical isomers ofsaid antisense-oligonucleotide.

Preferably the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceGGACGCGTAT (Seq. ID No. 7), and the antisense-oligonucleotide comprisesa sequence capable of hybridizing with said sequence GGACGCGTAT (Seq. IDNo. 7) and salts and optical isomers of said antisense-oligonucleotide.

Slightly reworded the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of theopen reading frame of the gene encoding the TGF-R_(II) or with a regionof the mRNA encoding the TGF-R_(II), wherein the region of the openreading frame of the gene encoding the TGF-R_(II) or the region of themRNA encoding the TGF-R_(II) comprises the sequence GGACGCGTAT (Seq. IDNo. 7), and the antisense-oligonucleotide comprises a sequence capableof hybridizing with said sequence GGACGCGTAT (Seq. ID No. 7) and saltsand optical isomers of said antisense-oligonucleotide.

Alternatively the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceGGACGCGTAT (Seq. ID No. 7), and the antisense-oligonucleotide comprisesa sequence complementary to the sequence GGACGCGTAT (Seq. ID No. 7) andsalts and optical isomers of said antisense-oligonucleotide.

Slightly reworded consisting of 10 to 28 nucleotides and at least two ofthe 10 to 28 nucleotides are LNAs and the antisense-oligonucleotide iscapable of hybridizing with a region of the open reading frame of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the open reading frame of the geneencoding the TGF-R_(II) or the region of the mRNA encoding theTGF-R_(II) comprises the sequence GGACGCGTAT (Seq. ID No. 7), and theantisense-oligonucleotide comprises a sequence complementary to thesequence GGACGCGTAT (Seq. ID No. 7) and salts and optical isomers ofsaid antisense-oligonucleotide.

Preferably the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceGTCTATGACG (Seq. ID No. 8), and the antisense-oligonucleotide comprisesa sequence capable of hybridizing with said sequence GTCTATGACG (Seq. IDNo. 8) and salts and optical isomers of said antisense-oligonucleotide.

Slightly reworded the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of theopen reading frame of the gene encoding the TGF-R_(II) or with a regionof the mRNA encoding the TGF-R_(II), wherein the region of the openreading frame of the gene encoding the TGF-R_(II) or the region of themRNA encoding the TGF-R_(II) comprises the sequence GTCTATGACG (Seq. IDNo. 8), and the antisense-oligonucleotide comprises a sequence capableof hybridizing with said sequence GTCTATGACG (Seq. ID No. 8) and saltsand optical isomers of said antisense-oligonucleotide.

Alternatively the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceGTCTATGACG (Seq. ID No. 8), and the antisense-oligonucleotide comprisesa sequence complementary to the sequence GTCTATGACG (Seq. ID No. 8) andsalts and optical isomers of said antisense-oligonucleotide.

Slightly reworded consisting of 10 to 28 nucleotides and at least two ofthe 10 to 28 nucleotides are LNAs and the antisense-oligonucleotide iscapable of hybridizing with a region of the open reading frame of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the open reading frame of the geneencoding the TGF-R_(II) or the region of the mRNA encoding theTGF-R_(II) comprises the sequence GTCTATGACG (Seq. ID No. 8), and theantisense-oligonucleotide comprises a sequence complementary to thesequence GTCTATGACG (Seq. ID No. 8) and salts and optical isomers ofsaid antisense-oligonucleotide.

Preferably the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceTTATTAATGC (Seq. ID No. 9), and the antisense-oligonucleotide comprisesa sequence capable of hybridizing with said sequence TTATTAATGC (Seq. IDNo. 9) and salts and optical isomers of said antisense-oligonucleotide.

Slightly reworded the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of theopen reading frame of the gene encoding the TGF-R_(II) or with a regionof the mRNA encoding the TGF-R_(II), wherein the region of the openreading frame of the gene encoding the TGF-R_(II) or the region of themRNA encoding the TGF-R_(II) comprises the sequence TTATTAATGC (Seq. IDNo. 9), and the antisense-oligonucleotide comprises a sequence capableof hybridizing with said sequence TTATTAATGC (Seq. ID No. 9) and saltsand optical isomers of said antisense-oligonucleotide.

Alternatively the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceTTATTAATGC (Seq. ID No. 9), and the antisense-oligonucleotide comprisesa sequence complementary to the sequence TTATTAATGC (Seq. ID No. 9) andsalts and optical isomers of said antisense-oligonucleotide.

Slightly reworded consisting of 10 to 28 nucleotides and at least two ofthe 10 to 28 nucleotides are LNAs and the antisense-oligonucleotide iscapable of hybridizing with a region of the open reading frame of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the open reading frame of the geneencoding the TGF-R_(II) or the region of the mRNA encoding theTGF-R_(II) comprises the sequence TTATTAATGC (Seq. ID No. 9), and theantisense-oligonucleotide comprises a sequence complementary to thesequence TTATTAATGC (Seq. ID No. 9) and salts and optical isomers ofsaid antisense-oligonucleotide.

The antisense-oligonucleotides of the present invention preferablycomprise 2 to 10 LNA units, more preferably 3 to 9 LNA units and stillmore preferably 4 to 8 LNA units and also preferably at least 6 non-LNAunits, more preferably at least 7 non-LNA units and most preferably atleast 8 non-LNA units. The non-LNA units are preferably DNA units. TheLNA units are preferably positioned at the 3′ terminal end (also named3′ terminus) and the 5′ terminal end (also named 5′ terminus).Preferably at least one and more preferably at least two LNA units arepresent at the 3′ terminal end and/or at the 5′ terminal end.

Thus, preferred are antisense-oligonucleotides of the present inventionwhich contain 3 to 10 LNA units and which especially contain 1 to 5 LNAunits at the 5′ terminal end and 1 to 5 LNA units at the 3′ terminal endof the antisense-oligonucleotide and between the LNA units at least 7and more preferably at least 8 DNA units.

Moreover, the antisense-oligonucleotides may contain common nucleobasessuch as adenine, guanine, cytosine, thymine and uracil as well as commonderivatives thereof. The antisense-oligonucleotides of the presentinvention may also contain modified internucleotide bridges such asphosphorothioate or phosphorodithioate instead of phosphate bridges.Such modifications may be present only in the LNA segments or only inthe non-LNA segment of the antisense-oligonucleotide.

Thus, the present invention is also directed toantisense-oligonucleotide(s) consisting of 12 to 24 nucleotides and atleast three of the 12 to 24 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceCTGGTCCATTC (Seq. ID No. 296), TGGTCCATTCA (Seq. ID No. 297),CTGGTCCATTCA (Seq. ID No. 298), TCCCTAAACAC (Seq. ID No. 299),CCCTAAACACT (Seq. ID No. 300), TCCCTAAACACT (Seq. ID No. 301),CACTACCAAAT (Seq. ID No. 302), ACTACCAAATA (Seq. ID No. 303),CACTACCAAATA (Seq. ID No. 304), TGGACGCGTAT (Seq. ID No. 305),GGACGCGTATC (Seq. ID No. 306), TGGACGCGTATC (Seq. ID No. 307),GGTCTATGACG (Seq. ID No. 308), GTCTATGACGA (Seq. ID No. 309),GGTCTATGACGA (Seq. ID No. 310), TTTATTAATGC (Seq. ID No. 311),TTATTAATGCC (Seq. ID No. 312), or TTTATTAATGCC (Seq. ID No. 313), andthe antisense-oligonucleotide comprises a sequence capable ofhybridizing with said sequence CTGGTCCATTC (Seq. ID No. 296),TGGTCCATTCA (Seq. ID No. 297), CTGGTCCATTCA (Seq. ID No. 298),TCCCTAAACAC (Seq. ID No. 299), CCCTAAACACT (Seq. ID No. 300),TCCCTAAACACT (Seq. ID No. 301), CACTACCAAAT (Seq. ID No. 302),ACTACCAAATA (Seq. ID No. 303), CACTACCAAATA (Seq. ID No. 304),TGGACGCGTAT (Seq. ID No. 305), GGACGCGTATC (Seq. ID No. 306),TGGACGCGTATC (Seq. ID No. 307), GGTCTATGACG (Seq. ID No. 308),GTCTATGACGA (Seq. ID No. 309), GGTCTATGACGA (Seq. ID No. 310),TTTATTAATGC (Seq. ID No. 311), TTATTAATGCC (Seq. ID No. 312), orTTTATTAATGCC (Seq. ID No. 313) respectively and salts and opticalisomers of said antisense-oligonucleotide.

Alternatively the present invention is directed toantisense-oligonucleotide(s) consisting of 12 to 24 nucleotides and atleast three of the 12 to 24 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceCTGGTCCATTC (Seq. ID No. 296), TGGTCCATTCA (Seq. ID No. 297),CTGGTCCATTCA (Seq. ID No. 298), TCCCTAAACAC (Seq. ID No. 299),CCCTAAACACT (Seq. ID No. 300), TCCCTAAACACT (Seq. ID No. 301),CACTACCAAAT (Seq. ID No. 302), ACTACCAAATA (Seq. ID No. 303),CACTACCAAATA (Seq. ID No. 304), TGGACGCGTAT (Seq. ID No. 305),GGACGCGTATC (Seq. ID No. 306), TGGACGCGTATC (Seq. ID No. 307),GGTCTATGACG (Seq. ID No. 308), GTCTATGACGA (Seq. ID No. 309),GGTCTATGACGA (Seq. ID No. 310), TTTATTAATGC (Seq. ID No. 311),TTATTAATGCC (Seq. ID No. 312), or TTTATTAATGCC (Seq. ID No. 313), andthe antisense-oligonucleotide comprises a sequence complementary to thesequence CTGGTCCATTC (Seq. ID No. 296), TGGTCCATTCA (Seq. ID No. 297),CTGGTCCATTCA (Seq. ID No. 298), TCCCTAAACAC (Seq. ID No. 299),CCCTAAACACT (Seq. ID No. 300), TCCCTAAACACT (Seq. ID No. 301),CACTACCAAAT (Seq. ID No. 302), ACTACCAAATA (Seq. ID No. 303),CACTACCAAATA (Seq. ID No. 304), TGGACGCGTAT (Seq. ID No. 305),GGACGCGTATC (Seq. ID No. 306), TGGACGCGTATC (Seq. ID No. 307),GGTCTATGACG (Seq. ID No. 308), GTCTATGACGA (Seq. ID No. 309),GGTCTATGACGA (Seq. ID No. 310), TTTATTAATGC (Seq. ID No. 311),TTATTAATGCC (Seq. ID No. 312), or TTTATTAATGCC (Seq. ID No. 313)respectively and salts and optical isomers of saidantisense-oligonucleotide.

Preferably, the present invention is also directed toantisense-oligonucleotide(s) consisting of 12 to 24 nucleotides and atleast three of the 12 to 24 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceCTGGTCCATTC (Seq. ID No. 296), TGGTCCATTCA (Seq. ID No. 297), orCTGGTCCATTCA (Seq. ID No. 298), and the antisense-oligonucleotidecomprises a sequence capable of hybridizing with said sequenceCTGGTCCATTC (Seq. ID No. 296), TGGTCCATTCA (Seq. ID No. 297), orCTGGTCCATTCA (Seq. ID No. 298) respectively and salts and opticalisomers of said antisense-oligonucleotide.

Slightly reworded, the present invention is also directed toantisense-oligonucleotide(s) consisting of 12 to 24 nucleotides and atleast three of the 12 to 24 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceCTGGTCCATTC (Seq. ID No. 296), TGGTCCATTCA (Seq. ID No. 297), orCTGGTCCATTCA (Seq. ID No. 298), and the antisense-oligonucleotidecomprises a sequence complementary to the sequence CTGGTCCATTC (Seq. IDNo. 296), TGGTCCATTCA (Seq. ID No. 297), or CTGGTCCATTCA (Seq. ID No.298) respectively and salts and optical isomers of saidantisense-oligonucleotide.

Preferably, the present invention is also directed toantisense-oligonucleotide(s) consisting of 12 to 24 nucleotides and atleast three of the 12 to 24 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceTCCCTAAACAC (Seq. ID No. 299), CCCTAAACACT (Seq. ID No. 300), orTCCCTAAACACT (Seq. ID No. 301), and the antisense-oligonucleotidecomprises a sequence capable of hybridizing with said sequenceTCCCTAAACAC (Seq. ID No. 299), CCCTAAACACT (Seq. ID No. 300), orTCCCTAAACACT (Seq. ID No. 301) respectively and salts and opticalisomers of said antisense-oligonucleotide.

Slightly reworded, the present invention is also directed toantisense-oligonucleotide(s) consisting of 12 to 24 nucleotides and atleast three of the 12 to 24 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceTCCCTAAACAC (Seq. ID No. 299), CCCTAAACACT (Seq. ID No. 300), orTCCCTAAACACT (Seq. ID No. 301), and the antisense-oligonucleotidecomprises a sequence complementary to the sequence TCCCTAAACAC (Seq. IDNo. 299), CCCTAAACACT (Seq. ID No. 300), or TCCCTAAACACT (Seq. ID No.301) respectively and salts and optical isomers of saidantisense-oligonucleotide.

Preferably, the present invention is also directed toantisense-oligonucleotide(s) consisting of 12 to 24 nucleotides and atleast three of the 12 to 24 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceCACTACCAAAT (Seq. ID No. 302), ACTACCAAATA (Seq. ID No. 303), orCACTACCAAATA (Seq. ID No. 304), and the antisense-oligonucleotidecomprises a sequence capable of hybridizing with said sequenceCACTACCAAAT (Seq. ID No. 302), ACTACCAAATA (Seq. ID No. 303), orCACTACCAAATA (Seq. ID No. 304) respectively and salts and opticalisomers of said antisense-oligonucleotide.

Slightly reworded, the present invention is also directed toantisense-oligonucleotide(s) consisting of 12 to 24 nucleotides and atleast three of the 12 to 24 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceCACTACCAAAT (Seq. ID No. 302), ACTACCAAATA (Seq. ID No. 303), orCACTACCAAATA (Seq. ID No. 304), and the antisense-oligonucleotidecomprises a sequence complementary to the sequence CACTACCAAAT (Seq. IDNo. 302), ACTACCAAATA (Seq. ID No. 303), or CACTACCAAATA (Seq. ID No.304) respectively and salts and optical isomers of saidantisense-oligonucleotide.

Preferably, the present invention is also directed toantisense-oligonucleotide(s) consisting of 12 to 24 nucleotides and atleast three of the 12 to 24 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceTGGACGCGTAT (Seq. ID No. 305), GGACGCGTATC (Seq. ID No. 306), orTGGACGCGTATC (Seq. ID No. 307), and the antisense-oligonucleotidecomprises a sequence capable of hybridizing with said sequenceTGGACGCGTAT (Seq. ID No. 305), GGACGCGTATC (Seq. ID No. 306), orTGGACGCGTATC (Seq. ID No. 307) respectively and salts and opticalisomers of said antisense-oligonucleotide.

Slightly reworded, the present invention is also directed toantisense-oligonucleotide(s) consisting of 12 to 24 nucleotides and atleast three of the 12 to 24 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceTGGACGCGTAT (Seq. ID No. 305), GGACGCGTATC (Seq. ID No. 306), orTGGACGCGTATC (Seq. ID No. 307), and the antisense-oligonucleotidecomprises a sequence complementary to the sequence TGGACGCGTAT (Seq. IDNo. 305), GGACGCGTATC (Seq. ID No. 306), or TGGACGCGTATC (Seq. ID No.307) respectively and salts and optical isomers of saidantisense-oligonucleotide.

Preferably, the present invention is also directed toantisense-oligonucleotide(s) consisting of 12 to 24 nucleotides and atleast three of the 12 to 24 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceGGTCTATGACG (Seq. ID No. 308), GTCTATGACGA (Seq. ID No. 309), orGGTCTATGACGA (Seq. ID No. 310), and the antisense-oligonucleotidecomprises a sequence capable of hybridizing with said sequenceGGTCTATGACG (Seq. ID No. 308), GTCTATGACGA (Seq. ID No. 309), orGGTCTATGACGA (Seq. ID No. 310) respectively and salts and opticalisomers of said antisense-oligonucleotide.

Slightly reworded, the present invention is also directed toantisense-oligonucleotide(s) consisting of 12 to 24 nucleotides and atleast three of the 12 to 24 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceGGTCTATGACG (Seq. ID No. 308), GTCTATGACGA (Seq. ID No. 309), orGGTCTATGACGA (Seq. ID No. 310), and the antisense-oligonucleotidecomprises a sequence complementary to the sequence GGTCTATGACG (Seq. IDNo. 308), GTCTATGACGA (Seq. ID No. 309), or GGTCTATGACGA (Seq. ID No.310) respectively and salts and optical isomers of saidantisense-oligonucleotide.

Preferably, the present invention is also directed toantisense-oligonucleotide(s) consisting of 12 to 24 nucleotides and atleast three of the 12 to 24 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceTTTATTAATGC (Seq. ID No. 311), TTATTAATGCC (Seq. ID No. 312), orTTTATTAATGCC (Seq. ID No. 313), and the antisense-oligonucleotidecomprises a sequence capable of hybridizing with said sequenceTTTATTAATGC (Seq. ID No. 311), TTATTAATGCC (Seq. ID No. 312), orTTTATTAATGCC (Seq. ID No. 313) respectively and salts and opticalisomers of said antisense-oligonucleotide.

Slightly reworded, the present invention is also directed toantisense-oligonucleotide(s) consisting of 12 to 24 nucleotides and atleast three of the 12 to 24 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceTTTATTAATGC (Seq. ID No. 311), TTATTAATGCC (Seq. ID No. 312), orTTTATTAATGCC (Seq. ID No. 313), and the antisense-oligonucleotidecomprises a sequence complementary to the sequence TTTATTAATGC (Seq. IDNo. 311), TTATTAATGCC (Seq. ID No. 312), or TTTATTAATGCC (Seq. ID No.313) respectively and salts and optical isomers of saidantisense-oligonucleotide.

The antisense-oligonucleotides of the present invention preferablycomprise 3 to 10 LNA units, more preferably 3 to 9 LNA units and stillmore preferably 4 to 8 LNA units and also preferably at least 6 non-LNAunits, more preferably at least 7 non-LNA units and most preferably atleast 8 non-LNA units. The non-LNA units are preferably DNA units. TheLNA units are preferably positioned at the 3′ terminal end (also named3′ terminus) and the 5′ terminal end (also named 5′ terminus).Preferably at least one and more preferably at least two LNA units arepresent at the 3′ terminal end and/or at the 5′ terminal end.

Thus, preferred are antisense-oligonucleotides of the present inventionwhich contain 3 to 10 LNA units and which especially contain 1 to 5 LNAunits at the 5′ terminal end and 1 to 5 LNA units at the 3′ terminal endof the antisense-oligonucleotide and between the LNA units at least 7and more preferably at least 8 DNA units.

Moreover, the antisense-oligonucleotides may contain common nucleobasessuch as adenine, guanine, cytosine, thymine and uracil as well as commonderivatives thereof. The antisense-oligonucleotides of the presentinvention may also contain modified internucleotide bridges such asphosphorothioate or phosphorodithioate instead of phosphate bridges.Such modifications may be present only in the LNA segments or only inthe non-LNA segment of the antisense-oligonucleotide.

Thus, the present invention is also directed toantisense-oligonucleotide(s) consisting of 14 to 20 more preferably 14to 18 nucleotides and at least four of the 14 to 20 more preferably 14to 18 nucleotides are LNAs and the antisense-oligonucleotide is capableof hybridizing with a region of the gene encoding the TGF-R_(II) or witha region of the mRNA encoding the TGF-R_(II), wherein the region of thegene encoding the TGF-R_(II) or the region of the mRNA encoding theTGF-R_(II) comprises the sequence ACTGGTCCATTC (Seq. ID No. 314),TGGTCCATTCAT (Seq. ID No. 315), CTGGTCCATTCAT (Seq. ID No. 316),ACTGGTCCATTCA (Seq. ID No. 317), ACTGGTCCATTCAT (Seq. ID No. 318),CTCCCTAAACAC (Seq. ID No. 319), CCCTAAACACTA (Seq. ID No. 320),TCCCTAAACACTA (Seq. ID No. 321), CTCCCTAAACACT (Seq. ID No. 322),CTCCCTAAACACTA (Seq. ID No. 323), ACACTACCAAAT (Seq. ID No. 324),ACTACCAAATAG (Seq. ID No. 325), CACTACCAAATAG (Seq. ID No. 326),ACACTACCAAATA (Seq. ID No. 327), ACACTACCAAATAG (Seq. ID No. 328),GTGGACGCGTAT (Seq. ID No. 329), GGACGCGTATCG (Seq. ID No. 330),TGGACGCGTATCG (Seq. ID No. 331), GTGGACGCGTATC (Seq. ID No. 332),GTGGACGCGTATCG (Seq. ID No. 333), CGGTCTATGACG (Seq. ID No. 334),GTCTATGACGAG (Seq. ID No. 335), GGTCTATGACGAG (Seq. ID No. 336),CGGTCTATGACGA (Seq. ID No. 337), CGGTCTATGACGAG (Seq. ID No. 338),CTTTATTAATGC (Seq. ID No. 339), TTATTAATGCCT (Seq. ID No. 340),TTTATTAATGCCT (Seq. ID No. 341), CTTTATTAATGCC (Seq. ID No. 342), orCTTTATTAATGCCT (Seq. ID No. 343), and the antisense-oligonucleotidecomprises a sequence capable of hybridizing with said sequenceACTGGTCCATTC (Seq. ID No. 314), TGGTCCATTCAT (Seq. ID No. 315),CTGGTCCATTCAT (Seq. ID No. 316), ACTGGTCCATTCA (Seq. ID No. 317),ACTGGTCCATTCAT (Seq. ID No. 318), CTCCCTAAACAC (Seq. ID No. 319),CCCTAAACACTA (Seq. ID No. 320), TCCCTAAACACTA (Seq. ID No. 321),CTCCCTAAACACT (Seq. ID No. 322), CTCCCTAAACACTA (Seq. ID No. 323),ACACTACCAAAT (Seq. ID No. 324), ACTACCAAATAG (Seq. ID No. 325),CACTACCAAATAG (Seq. ID No. 326), ACACTACCAAATA (Seq. ID No. 327),ACACTACCAAATAG (Seq. ID No. 328), GTGGACGCGTAT (Seq. ID No. 329),GGACGCGTATCG (Seq. ID No. 330), TGGACGCGTATCG (Seq. ID No. 331),GTGGACGCGTATC (Seq. ID No. 332), GTGGACGCGTATCG (Seq. ID No. 333),CGGTCTATGACG (Seq. ID No. 334), GTCTATGACGAG (Seq. ID No. 335),GGTCTATGACGAG (Seq. ID No. 336), CGGTCTATGACGA (Seq. ID No. 337),CGGTCTATGACGAG (Seq. ID No. 338), CTTTATTAATGC (Seq. ID No. 339),TTATTAATGCCT (Seq. ID No. 340), TTTATTAATGCCT (Seq. ID No. 341),CTTTATTAATGCC (Seq. ID No. 342), or CTTTATTAATGCCT (Seq. ID No. 343),respectively and salts and optical isomers of saidantisense-oligonucleotide.

Alternatively the present invention is directed toantisense-oligonucleotide(s) consisting of 14 to 20 more preferably 14to 18 nucleotides and at least four of the 14 to 20 more preferably 14to 18 nucleotides are LNAs and the antisense-oligonucleotide is capableof hybridizing with a region of the gene encoding the TGF-R_(II) or witha region of the mRNA encoding the TGF-R_(II), wherein the region of thegene encoding the TGF-R_(II) or the region of the mRNA encoding theTGF-R_(II) comprises the sequence ACTGGTCCATTC (Seq. ID No. 314),TGGTCCATTCAT (Seq. ID No. 315), CTGGTCCATTCAT (Seq. ID No. 316),ACTGGTCCATTCA (Seq. ID No. 317), ACTGGTCCATTCAT (Seq. ID No. 318),CTCCCTAAACAC (Seq. ID No. 319), CCCTAAACACTA (Seq. ID No. 320),TCCCTAAACACTA (Seq. ID No. 321), CTCCCTAAACACT (Seq. ID No. 322),CTCCCTAAACACTA (Seq. ID No. 323), ACACTACCAAAT (Seq. ID No. 324),ACTACCAAATAG (Seq. ID No. 325), CACTACCAAATAG (Seq. ID No. 326),ACACTACCAAATA (Seq. ID No. 327), ACACTACCAAATAG (Seq. ID No. 328),GTGGACGCGTAT (Seq. ID No. 329), GGACGCGTATCG (Seq. ID No. 330),TGGACGCGTATCG (Seq. ID No. 331), GTGGACGCGTATC (Seq. ID No. 332),GTGGACGCGTATCG (Seq. ID No. 333), CGGTCTATGACG (Seq. ID No. 334),GTCTATGACGAG (Seq. ID No. 335), GGTCTATGACGAG (Seq. ID No. 336),CGGTCTATGACGA (Seq. ID No. 337), CGGTCTATGACGAG (Seq. ID No. 338),CTTTATTAATGC (Seq. ID No. 339), TTATTAATGCCT (Seq. ID No. 340),TTTATTAATGCCT (Seq. ID No. 341), CTTTATTAATGCC (Seq. ID No. 342), orCTTTATTAATGCCT (Seq. ID No. 343), and the antisense-oligonucleotidecomprises a sequence complementary to the sequence ACTGGTCCATTC (Seq. IDNo. 314), TGGTCCATTCAT (Seq. ID No. 315), CTGGTCCATTCAT (Seq. ID No.316), ACTGGTCCATTCA (Seq. ID No. 317), ACTGGTCCATTCAT (Seq. ID No. 318),CTCCCTAAACAC (Seq. ID No. 319), CCCTAAACACTA (Seq. ID No. 320),TCCCTAAACACTA (Seq. ID No. 321), CTCCCTAAACACT (Seq. ID No. 322),CTCCCTAAACACTA (Seq. ID No. 323), ACACTACCAAAT (Seq. ID No. 324),ACTACCAAATAG (Seq. ID No. 325), CACTACCAAATAG (Seq. ID No. 326),ACACTACCAAATA (Seq. ID No. 327), ACACTACCAAATAG (Seq. ID No. 328),GTGGACGCGTAT (Seq. ID No. 329), GGACGCGTATCG (Seq. ID No. 330),TGGACGCGTATCG (Seq. ID No. 331), GTGGACGCGTATC (Seq. ID No. 332),GTGGACGCGTATCG (Seq. ID No. 333), CGGTCTATGACG (Seq. ID No. 334),GTCTATGACGAG (Seq. ID No. 335), GGTCTATGACGAG (Seq. ID No. 336),CGGTCTATGACGA (Seq. ID No. 337), CGGTCTATGACGAG (Seq. ID No. 338),CTTTATTAATGC (Seq. ID No. 339), TTATTAATGCCT (Seq. ID No. 340),TTTATTAATGCCT (Seq. ID No. 341), CTTTATTAATGCC (Seq. ID No. 342), orCTTTATTAATGCCT (Seq. ID No. 343), respectively and salts and opticalisomers of said antisense-oligonucleotide.

Preferably the present invention is also directed toantisense-oligonucleotide(s) consisting of 14 to 20 more preferably 14to 18 nucleotides and at least four of the 14 to 20 more preferably 14to 18 nucleotides are LNAs and the antisense-oligonucleotide is capableof hybridizing with a region of the gene encoding the TGF-R_(II) or witha region of the mRNA encoding the TGF-R_(II), wherein the region of thegene encoding the TGF-R_(II) or the region of the mRNA encoding theTGF-R_(II) comprises the sequence ACTGGTCCATTC (Seq. ID No. 314),TGGTCCATTCAT (Seq. ID No. 315), CTGGTCCATTCAT (Seq. ID No. 316),ACTGGTCCATTCA (Seq. ID No. 317), ACTGGTCCATTCAT (Seq. ID No. 318), andthe antisense-oligonucleotide comprises a sequence capable ofhybridizing with said sequence ACTGGTCCATTC (Seq. ID No. 314),TGGTCCATTCAT (Seq. ID No. 315), CTGGTCCATTCAT (Seq. ID No. 316),ACTGGTCCATTCA (Seq. ID No. 317), ACTGGTCCATTCAT (Seq. ID No. 318),respectively and salts and optical isomers of saidantisense-oligonucleotide.

Slightly reworded, the present invention is directed toantisense-oligonucleotide(s) consisting of 14 to 20 more preferably 14to 18 nucleotides and at least four of the 14 to 20 more preferably 14to 18 nucleotides are LNAs and the antisense-oligonucleotide is capableof hybridizing with a region of the gene encoding the TGF-R_(II) or witha region of the mRNA encoding the TGF-R_(II), wherein the region of thegene encoding the TGF-R_(II) or the region of the mRNA encoding theTGF-R_(II) comprises the sequence ACTGGTCCATTC (Seq. ID No. 314),TGGTCCATTCAT (Seq. ID No. 315), CTGGTCCATTCAT (Seq. ID No. 316),ACTGGTCCATTCA (Seq. ID No. 317), or ACTGGTCCATTCAT (Seq. ID No. 318),and the antisense-oligonucleotide comprises a sequence complementary tothe sequence ACTGGTCCATTC (Seq. ID No. 314), TGGTCCATTCAT (Seq. ID No.315), CTGGTCCATTCAT (Seq. ID No. 316), ACTGGTCCATTCA (Seq. ID No. 317),or ACTGGTCCATTCAT (Seq. ID No. 318), respectively and salts and opticalisomers of said antisense-oligonucleotide.

Preferably the present invention is also directed toantisense-oligonucleotide(s) consisting of 14 to 20 more preferably 14to 18 nucleotides and at least four of the 14 to 20 more preferably 14to 18 nucleotides are LNAs and the antisense-oligonucleotide is capableof hybridizing with a region of the gene encoding the TGF-R_(II) or witha region of the mRNA encoding the TGF-R_(II), wherein the region of thegene encoding the TGF-R_(II) or the region of the mRNA encoding theTGF-R_(II) comprises the sequence CTCCCTAAACAC (Seq. ID No. 319),CCCTAAACACTA (Seq. ID No. 320), TCCCTAAACACTA (Seq. ID No. 321),CTCCCTAAACACT (Seq. ID No. 322), or CTCCCTAAACACTA (Seq. ID No. 323),and the antisense-oligonucleotide comprises a sequence capable ofhybridizing with said sequence CTCCCTAAACAC (Seq. ID No. 319),CCCTAAACACTA (Seq. ID No. 320), TCCCTAAACACTA (Seq. ID No. 321),CTCCCTAAACACT (Seq. ID No. 322), or CTCCCTAAACACTA (Seq. ID No. 323),respectively and salts and optical isomers of saidantisense-oligonucleotide.

Slightly reworded, the present invention is directed toantisense-oligonucleotide(s) consisting of 14 to 20 more preferably 14to 18 nucleotides and at least four of the 14 to 20 more preferably 14to 18 nucleotides are LNAs and the antisense-oligonucleotide is capableof hybridizing with a region of the gene encoding the TGF-R_(II) or witha region of the mRNA encoding the TGF-R_(II), wherein the region of thegene encoding the TGF-R_(II) or the region of the mRNA encoding theTGF-R_(II) comprises the sequence CTCCCTAAACAC (Seq. ID No. 319),CCCTAAACACTA (Seq. ID No. 320), TCCCTAAACACTA (Seq. ID No. 321),CTCCCTAAACACT (Seq. ID No. 322), or CTCCCTAAACACTA (Seq. ID No. 323),and the antisense-oligonucleotide comprises a sequence complementary tothe sequence CTCCCTAAACAC (Seq. ID No. 319), CCCTAAACACTA (Seq. ID No.320), TCCCTAAACACTA (Seq. ID No. 321), CTCCCTAAACACT (Seq. ID No. 322),or CTCCCTAAACACTA (Seq. ID No. 323), respectively and salts and opticalisomers of said antisense-oligonucleotide.

Preferably the present invention is also directed toantisense-oligonucleotide(s) consisting of 14 to 20 more preferably 14to 18 nucleotides and at least four of the 14 to 20 more preferably 14to 18 nucleotides are LNAs and the antisense-oligonucleotide is capableof hybridizing with a region of the gene encoding the TGF-R_(II) or witha region of the mRNA encoding the TGF-R_(II), wherein the region of thegene encoding the TGF-R_(II) or the region of the mRNA encoding theTGF-R_(II) comprises the sequence ACACTACCAAAT (Seq. ID No. 324),ACTACCAAATAG (Seq. ID No. 325), CACTACCAAATAG (Seq. ID No. 326),ACACTACCAAATA (Seq. ID No. 327), or ACACTACCAAATAG (Seq. ID No. 328),and the antisense-oligonucleotide comprises a sequence capable ofhybridizing with said sequence ACACTACCAAAT (Seq. ID No. 324),ACTACCAAATAG (Seq. ID No. 325), CACTACCAAATAG (Seq. ID No. 326),ACACTACCAAATA (Seq. ID No. 327), or ACACTACCAAATAG (Seq. ID No. 328),respectively and salts and optical isomers of saidantisense-oligonucleotide.

Slightly reworded, the present invention is directed toantisense-oligonucleotide(s) consisting of 14 to 20 more preferably 14to 18 nucleotides and at least four of the 14 to 20 more preferably 14to 18 nucleotides are LNAs and the antisense-oligonucleotide is capableof hybridizing with a region of the gene encoding the TGF-R_(II) or witha region of the mRNA encoding the TGF-R_(II), wherein the region of thegene encoding the TGF-R_(II) or the region of the mRNA encoding theTGF-R_(II) comprises the sequence ACACTACCAAAT (Seq. ID No. 324),ACTACCAAATAG (Seq. ID No. 325), CACTACCAAATAG (Seq. ID No. 326),ACACTACCAAATA (Seq. ID No. 327), or ACACTACCAAATAG (Seq. ID No. 328),and the antisense-oligonucleotide comprises a sequence complementary tothe sequence ACACTACCAAAT (Seq. ID No. 324), ACTACCAAATAG (Seq. ID No.325), CACTACCAAATAG (Seq. ID No. 326), ACACTACCAAATA (Seq. ID No. 327),or ACACTACCAAATAG (Seq. ID No. 328), respectively and salts and opticalisomers of said antisense-oligonucleotide.

Preferably the present invention is also directed toantisense-oligonucleotide(s) consisting of 14 to 20 more preferably 14to 18 nucleotides and at least four of the 14 to 20 more preferably 14to 18 nucleotides are LNAs and the antisense-oligonucleotide is capableof hybridizing with a region of the gene encoding the TGF-R_(II) or witha region of the mRNA encoding the TGF-R_(II), wherein the region of thegene encoding the TGF-R_(II) or the region of the mRNA encoding theTGF-R_(II) comprises the sequence GTGGACGCGTAT (Seq. ID No. 329),GGACGCGTATCG (Seq. ID No. 330), TGGACGCGTATCG (Seq. ID No. 331),GTGGACGCGTATC (Seq. ID No. 332), or GTGGACGCGTATCG (Seq. ID No. 333),and the antisense-oligonucleotide comprises a sequence capable ofhybridizing with said sequence GTGGACGCGTAT (Seq. ID No. 329),GGACGCGTATCG (Seq. ID No. 330), TGGACGCGTATCG (Seq. ID No. 331),GTGGACGCGTATC (Seq. ID No. 332), or GTGGACGCGTATCG (Seq. ID No. 333),respectively and salts and optical isomers of saidantisense-oligonucleotide.

Slightly reworded, the present invention is directed toantisense-oligonucleotide(s) consisting of 14 to 20 more preferably 14to 18 nucleotides and at least four of the 14 to 20 more preferably 14to 18 nucleotides are LNAs and the antisense-oligonucleotide is capableof hybridizing with a region of the gene encoding the TGF-R_(II) or witha region of the mRNA encoding the TGF-R_(II), wherein the region of thegene encoding the TGF-R_(II) or the region of the mRNA encoding theTGF-R_(II) comprises the sequence GTGGACGCGTAT (Seq. ID No. 329),GGACGCGTATCG (Seq. ID No. 330), TGGACGCGTATCG (Seq. ID No. 331),GTGGACGCGTATC (Seq. ID No. 332), or GTGGACGCGTATCG (Seq. ID No. 333),and the antisense-oligonucleotide comprises a sequence complementary tothe sequence GTGGACGCGTAT (Seq. ID No. 329), GGACGCGTATCG (Seq. ID No.330), TGGACGCGTATCG (Seq. ID No. 331), GTGGACGCGTATC (Seq. ID No. 332),or GTGGACGCGTATCG (Seq. ID No. 333), respectively and salts and opticalisomers of said antisense-oligonucleotide.

Preferably the present invention is also directed toantisense-oligonucleotide(s) consisting of 14 to 20 more preferably 14to 18 nucleotides and at least four of the 14 to 20 more preferably 14to 18 nucleotides are LNAs and the antisense-oligonucleotide is capableof hybridizing with a region of the gene encoding the TGF-R_(II) or witha region of the mRNA encoding the TGF-R_(II), wherein the region of thegene encoding the TGF-R_(II) or the region of the mRNA encoding theTGF-R_(II) comprises the sequence CGGTCTATGACG (Seq. ID No. 334),GTCTATGACGAG (Seq. ID No. 335), GGTCTATGACGAG (Seq. ID No. 336),CGGTCTATGACGA (Seq. ID No. 337), or CGGTCTATGACGAG (Seq. ID No. 338),and the antisense-oligonucleotide comprises a sequence capable ofhybridizing with said sequence CGGTCTATGACG (Seq. ID No. 334),GTCTATGACGAG (Seq. ID No. 335), GGTCTATGACGAG (Seq. ID No. 336),CGGTCTATGACGA (Seq. ID No. 337), or CGGTCTATGACGAG (Seq. ID No. 338),respectively and salts and optical isomers of saidantisense-oligonucleotide.

Slightly reworded, the present invention is directed toantisense-oligonucleotide(s) consisting of 14 to 20 more preferably 14to 18 nucleotides and at least four of the 14 to 20 more preferably 14to 18 nucleotides are LNAs and the antisense-oligonucleotide is capableof hybridizing with a region of the gene encoding the TGF-R_(II) or witha region of the mRNA encoding the TGF-R_(II), wherein the region of thegene encoding the TGF-R_(II) or the region of the mRNA encoding theTGF-R_(II) comprises the sequence CGGTCTATGACG (Seq. ID No. 334),GTCTATGACGAG (Seq. ID No. 335), GGTCTATGACGAG (Seq. ID No. 336),CGGTCTATGACGA (Seq. ID No. 337), or CGGTCTATGACGAG (Seq. ID No. 338),and the antisense-oligonucleotide comprises a sequence complementary tothe sequence CGGTCTATGACG (Seq. ID No. 334), GTCTATGACGAG (Seq. ID No.335), GGTCTATGACGAG (Seq. ID No. 336), CGGTCTATGACGA (Seq. ID No. 337),or CGGTCTATGACGAG (Seq. ID No. 338), respectively and salts and opticalisomers of said antisense-oligonucleotide.

Preferably the present invention is also directed toantisense-oligonucleotide(s) consisting of 14 to 20 more preferably 14to 18 nucleotides and at least four of the 14 to 20 more preferably 14to 18 nucleotides are LNAs and the antisense-oligonucleotide is capableof hybridizing with a region of the gene encoding the TGF-R_(II) or witha region of the mRNA encoding the TGF-R_(II), wherein the region of thegene encoding the TGF-R_(II) or the region of the mRNA encoding theTGF-R_(II) comprises the sequence CTTTATTAATGC (Seq. ID No. 339),TTATTAATGCCT (Seq. ID No. 340), TTTATTAATGCCT (Seq. ID No. 341),CTTTATTAATGCC (Seq. ID No. 342), or CTTTATTAATGCCT (Seq. ID No. 343),and the antisense-oligonucleotide comprises a sequence capable ofhybridizing with said sequence CTTTATTAATGC (Seq. ID No. 339),TTATTAATGCCT (Seq. ID No. 340), TTTATTAATGCCT (Seq. ID No. 341),CTTTATTAATGCC (Seq. ID No. 342), or CTTTATTAATGCCT (Seq. ID No. 343),respectively and salts and optical isomers of saidantisense-oligonucleotide.

Slightly reworded, the present invention is directed toantisense-oligonucleotide(s) consisting of 14 to 20 more preferably 14to 18 nucleotides and at least four of the 14 to 20 more preferably 14to 18 nucleotides are LNAs and the antisense-oligonucleotide is capableof hybridizing with a region of the gene encoding the TGF-R_(II) or witha region of the mRNA encoding the TGF-R_(II), wherein the region of thegene encoding the TGF-R_(II) or the region of the mRNA encoding theTGF-R_(II) comprises the sequence CTTTATTAATGC (Seq. ID No. 339),TTATTAATGCCT (Seq. ID No. 340), TTTATTAATGCCT (Seq. ID No. 341),CTTTATTAATGCC (Seq. ID No. 342), or CTTTATTAATGCCT (Seq. ID No. 343),and the antisense-oligonucleotide comprises a sequence complementary tothe sequence CTTTATTAATGC (Seq. ID No. 339), TTATTAATGCCT (Seq. ID No.340), TTTATTAATGCCT (Seq. ID No. 341), CTTTATTAATGCC (Seq. ID No. 342),or CTTTATTAATGCCT (Seq. ID No. 343), respectively and salts and opticalisomers of said antisense-oligonucleotide.

The antisense-oligonucleotides of the present invention preferablycomprise 4 to 11 LNA units, more preferably 4 to 10 LNA units and stillmore preferably 4 to 8 LNA units and also preferably at least 6 non-LNAunits, more preferably at least 7 non-LNA units and most preferably atleast 8 non-LNA units. The non-LNA units are preferably DNA units. TheLNA units are preferably positioned at the 3′ terminal end (also named3′ terminus) and the 5′ terminal end (also named 5′ terminus).Preferably at least one and more preferably at least two LNA units arepresent at the 3′ terminal end and/or at the 5′ terminal end.

Thus, preferred are antisense-oligonucleotides of the present inventionwhich contain 3 to 10 LNA units and which especially contain 1 to 5 LNAunits at the 5′ terminal end and 1 to 5 LNA units at the 3′ terminal endof the antisense-oligonucleotide and between the LNA units at least 7and more preferably at least 8 DNA units.

Moreover, the antisense-oligonucleotides may contain common nucleobasessuch as adenine, guanine, cytosine, thymine and uracil as well as commonderivatives thereof. The antisense-oligonucleotides of the presentinvention may also contain modified internucleotide bridges such asphosphorothioate or phosphorodithioate instead of phosphate bridges.Such modifications may be present only in the LNA segments or only inthe non-LNA segment of the antisense-oligonucleotide.

Thus, the present invention is directed to antisense-oligonucleotide(s)consisting of 10 to 28 nucleotides and at least two of the 10 to 28nucleotides are LNAs and the antisense-oligonucleotide is capable ofhybridizing with a region of the gene encoding the TGF-R_(II) or with aregion of the mRNA encoding the TGF-R_(II), wherein theantisense-oligonucleotide is represented by the following sequence5′-N¹-GTCATAGA-N²-3′ (Seq. ID No. 12) or 5′-N³-ACGCGTCC-N⁴-3′ (Seq. IDNo. 98) or 5′-N¹¹-TGTTTAGG-N¹²-3′ (Seq. ID No. 10) or5′-N⁵-TTTGGTAG-N⁶-3′ (Seq. ID No. 11) or 5′-N⁷-AATGGACC-N⁸-3′ (Seq. IDNo. 100) or 5′-N⁹-ATTAATAA-N¹⁰-3′ (Seq. ID No. 101), wherein

N¹ represents: CATGGCAGACCCCGCTGCTC- (Seq. ID No. 509),ATGGCAGACCCCGCTGCTC- (Seq. ID No. 510), TGGCAGACCCCGCTGCTC- (Seq. ID No.511), GGCAGACCCCGCTGCTC- (Seq. ID No. 512), GCAGACCCCGCTGCTC- (Seq. IDNo. 513), CAGACCCCGCTGCTC- (Seq. ID No. 514), AGACCCCGCTGCTC- (Seq. IDNo. 515), GACCCCGCTGCTC- (Seq. ID No. 516), ACCCCGCTGCTC- (Seq. ID No.517), CCCCGCTGCTC- (Seq. ID No. 518), CCCGCTGCTC- (Seq. ID No. 519),CCGCTGCTC-, CGCTGCTC-, GCTGCTC-, CTGCTC-, TGCTC-, GCTC-, CTC-, TC-, orC-;N² represents: -C, -CC, -CCG, -CCGA, -CCGAG, -CCGAGC, -CCGAGCC,-CCGAGCCC, -CCGAGCCCC, -CCGAGCCCCC (Seq. ID No. 520), -CCGAGCCCCCA (Seq.ID No. 521), -CCGAGCCCCCAG (Seq. ID No. 522), -CCGAGCCCCCAGC (Seq. IDNo. 523), -CCGAGCCCCCAGCG (Seq. ID No. 524), -CCGAGCCCCCAGCGC (Seq. IDNo. 525), -CCGAGCCCCCAGCGCA (Seq. ID No. 526), -CCGAGCCCCCAGCGCAG (Seq.ID No. 527), -CCGAGCCCCCAGCGCAGC (Seq. ID No. 528), -CCGAGCCCCCAGCGCAGCG(Seq. ID No. 529), or -CCGAGCCCCCAGCGCAGCGG (Seq. ID No. 530);N³ represents: GGTGGGATCGTGCTGGCGAT- (Seq. ID No. 531),GTGGGATCGTGCTGGCGAT- (Seq. ID No. 532), TGGGATCGTGCTGGCGAT- (Seq. ID No.533), GGGATCGTGCTGGCGAT- (Seq. ID No. 534), GGATCGTGCTGGCGAT- (Seq. IDNo. 535), GATCGTGCTGGCGAT- (Seq. ID No. 536), ATCGTGCTGGCGAT- (Seq. IDNo. 537), TCGTGCTGGCGAT- (Seq. ID No. 538), CGTGCTGGCGAT- (Seq. ID No.539), GTGCTGGCGAT- (Seq. ID No. 540), TGCTGGCGAT- (Seq. ID No. 541),GCTGGCGAT-, CTGGCGAT-, TGGCGAT-, GGCGAT-, GCGAT-, CGAT-, GAT-, AT-, orT-;N⁴ represents: -ACAGGACGATGTGCAGCGGC (Seq. ID No. 542),-ACAGGACGATGTGCAGCGG (Seq. ID No. 543), -ACAGGACGATGTGCAGCG (Seq. ID No.544), -ACAGGACGATGTGCAGC (Seq. ID No. 545), -ACAGGACGATGTGCAG (Seq. IDNo. 546), -ACAGGACGATGTGCA (Seq. ID No. 547), -ACAGGACGATGTGC (Seq. IDNo. 548), -ACAGGACGATGTG (Seq. ID No. 549), -ACAGGACGATGT (Seq. ID No.550), -ACAGGACGATG (Seq. ID No. 551), -ACAGGACGAT (Seq. ID No. 552),-ACAGGACGA, -ACAGGACG, -ACAGGAC, -ACAGGA, -ACAGG, -ACAG, -ACA, -AC, or-A;N⁵ represents: GCCCAGCCTGCCCCAGAAGAGCTA- (Seq. ID No. 553),CCCAGCCTGCCCCAGAAGAGCTA- (Seq. ID No. 554), CCAGCCTGCCCCAGAAGAGCTA-(Seq. ID No. 555), CAGCCTGCCCCAGAAGAGCTA- (Seq. ID No. 556),AGCCTGCCCCAGAAGAGCTA- (Seq. ID No. 557), GCCTGCCCCAGAAGAGCTA- (Seq. IDNo. 558), CCTGCCCCAGAAGAGCTA- (Seq. ID No. 559), CTGCCCCAGAAGAGCTA-(Seq. ID No. 560), TGCCCCAGAAGAGCTA- (Seq. ID No. 561), GCCCCAGAAGAGCTA-(Seq. ID No. 562), CCCCAGAAGAGCTA- (Seq. ID No. 563), CCCAGAAGAGCTA-(Seq. ID No. 564), CCAGAAGAGCTA- (Seq. ID No. 565), CAGAAGAGCTA- (Seq.ID No. 566), AGAAGAGCTA- (Seq. ID No. 567), GAAGAGCTA-, AAGAGCTA-,AGAGCTA-, GAGCTA-, AGCTA-, GCTA-, CTA-, TA-, or A-;N⁶ represents: -TGTTTAGGGAGCCGTCTTCAGGAA (Seq. ID No. 568),-TGTTTAGGGAGCCGTCTTCAGGA (Seq. ID No. 569), TGTTTAGGGAGCCGTCTTCAGG (Seq.ID No. 570), -TGTTTAGGGAGCCGTCTTCAG (Seq. ID No. 571),-TGTTTAGGGAGCCGTCTTCA (Seq. ID No. 572), -TGTTTAGGGAGCCGTCTTC (Seq. IDNo. 573), -TGTTTAGGGAGCCGTCTT (Seq. ID No. 574), -TGTTTAGGGAGCCGTCT(Seq. ID No. 575), -TGTTTAGGGAGCCGTC (Seq. ID No. 576), -TGTTTAGGGAGCCGT(Seq. ID No. 577), -TGTTTAGGGAGCCG (Seq. ID No. 578), -TGTTTAGGGAGCC(Seq. ID No. 579), -TGTTTAGGGAGC (Seq. ID No. 580), -TGTTTAGGGAG (Seq.ID No. 581), -TGTTTAGGGA (Seq. ID No. 582), -TGTTTAGGG, -TGTTTAGG,-TGTTTAG, -TGTTTA, -TGTTT, -TGTT, -TGT, -TG, or -T;N⁷ represents: TGAATCTTGAATATCTCATG- (Seq. ID No. 583),GAATCTTGAATATCTCATG- (Seq. ID No. 584), AATCTTGAATATCTCATG- (Seq. ID No.585), ATCTTGAATATCTCATG- (Seq. ID No. 586), TCTTGAATATCTCATG- (Seq. IDNo. 587), CTTGAATATCTCATG- (Seq. ID No. 588), TTGAATATCTCATG- (Seq. IDNo. 589), TGAATATCTCATG- (Seq. ID No. 590), GAATATCTCATG- (Seq. ID No.591), AATATCTCATG- (Seq. ID No. 592), ATATCTCATG- (Seq. ID No. 593),TATCTCATG-, ATCTCATG-, TCTCATG-, CTCATG-, TCATG-, CATG-, ATG-, TG-, orG-;N⁸ represents: -AGTATTCTAGAAACTCACCA (Seq. ID No. 594),-AGTATTCTAGAAACTCACC (Seq. ID No. 595), -AGTATTCTAGAAACTCAC (Seq. ID No.596), -AGTATTCTAGAAACTCA (Seq. ID No. 597), -AGTATTCTAGAAACTC (Seq. IDNo. 598), -AGTATTCTAGAAACT (Seq. ID No. 599), -AGTATTCTAGAAAC (Seq. IDNo. 600), -AGTATTCTAGAAA (Seq. ID No. 601), -AGTATTCTAGAA (Seq. ID No.602), -AGTATTCTAGA (Seq. ID No. 603), -AGTATTCTAG (Seq. ID No. 604),-AGTATTCTA, -AGTATTCT, -AGTATTC, -AGTATT, -AGTAT, -AGTA, -AGT, -AG, or-A;N⁹ represents: ATTCATATTTATATACAGGC- (Seq. ID No. 605),TTCATATTTATATACAGGC- (Seq. ID No. 606), TCATATTTATATACAGGC- (Seq. ID No.607), CATATTTATATACAGGC- (Seq. ID No. 608), ATATTTATATACAGGC- (Seq. IDNo. 609), TATTTATATACAGGC- (Seq. ID No. 610), ATTTATATACAGGC- (Seq. IDNo. 611), TTTATATACAGGC- (Seq. ID No. 612), TTATATACAGGC- (Seq. ID No.613), TATATACAGGC- (Seq. ID No. 614), ATATACAGGC- (Seq. ID No. 615),TATACAGGC-, ATACAGGC-, TACAGGC-, ACAGGC-, CAGGC-, AGGC-, GGC-, GC-, orC-;N¹⁰ represents: -AGTGCAAATGTTATTGGCTA (Seq. ID No. 616),-AGTGCAAATGTTATTGGCT (Seq. ID No. 617), -AGTGCAAATGTTATTGGC (Seq. ID No.618), -AGTGCAAATGTTATTGG (Seq. ID No. 619), -AGTGCAAATGTTATTG (Seq. IDNo. 620), -AGTGCAAATGTTATT (Seq. ID No. 621), -AGTGCAAATGTTAT (Seq. IDNo. 622), -AGTGCAAATGTTA (Seq. ID No. 623), -AGTGCAAATGTT (Seq. ID No.624), -AGTGCAAATGT (Seq. ID No. 625), -AGTGCAAATG (Seq. ID No. 626),-AGTGCAAAT, -AGTGCAAA, -AGTGCAA, -AGTGCA, -AGTGC, -AGTG, -AGT, -AG, or-A;N¹¹ represents: TGCCCCAGAAGAGCTATTTGGTAG- (Seq. ID No. 627),GCCCCAGAAGAGCTATTTGGTAG- (Seq. ID No. 628), CCCCAGAAGAGCTATTTGGTAG-(Seq. ID No. 629), CCCAGAAGAGCTATTTGGTAG- (Seq. ID No. 630),CCAGAAGAGCTATTTGGTAG- (Seq. ID No. 631), CAGAAGAGCTATTTGGTAG- (Seq. IDNo. 632), AGAAGAGCTATTTGGTAG- (Seq. ID No. 633), GAAGAGCTATTTGGTAG-(Seq. ID No. 634), AAGAGCTATTTGGTAG- (Seq. ID No. 635), AGAGCTATTTGGTAG-(Seq. ID No. 636), GAGCTATTTGGTAG- (Seq. ID No. 637), AGCTATTTGGTAG-(Seq. ID No. 638), GCTATTTGGTAG- (Seq. ID No. 639), CTATTTGGTAG- (Seq.ID No. 640), TATTTGGTAG- (Seq. ID No. 641), ATTTGGTAG-, TTTGGTAG-,TTGGTAG-, TGGTAG-, GGTAG-, GTAG-, TAG-, AG- or G-,N¹² represents: -GAGCCGTCTTCAGGAATCTTCTCC (Seq. ID No. 642),-GAGCCGTCTTCAGGAATCTTCTC (Seq. ID No. 643), GAGCCGTCTTCAGGAATCTTCT (Seq.ID No. 644), -GAGCCGTCTTCAGGAATCTTC (Seq. ID No. 645),-GAGCCGTCTTCAGGAATCTT (Seq. ID No. 646), -GAGCCGTCTTCAGGAATCT (Seq. IDNo. 647), -GAGCCGTCTTCAGGAATC (Seq. ID No. 648), -GAGCCGTCTTCAGGAAT(Seq. ID No. 649), -GAGCCGTCTTCAGGAA (Seq. ID No. 650), -GAGCCGTCTTCAGGA(Seq. ID No. 651), -GAGCCGTCTTCAGG (Seq. ID No. 652), -GAGCCGTCTTCAG(Seq. ID No. 653), -GAGCCGTCTTCA (Seq. ID No. 654), -GAGCCGTCTTC (Seq.ID No. 655), -GAGCCGTCTT (Seq. ID No. 656), -GAGCCGTCT, -GAGCCGTC,-GAGCCGT, -GAGCCG, -GAGCC, -GAGC, -GAG, -GA, or -G;and salts and optical isomers of the antisense-oligonucleotide.

Thus, the present invention is directed to antisense-oligonucleotide(s)consisting of 10 to 28 nucleotides and at least two of the 10 to 28nucleotides are LNAs and the antisense-oligonucleotide is capable ofhybridizing with a region of the gene encoding the TGF-R_(II) or with aregion of the mRNA encoding the TGF-R_(II), wherein theantisense-oligonucleotide is represented by the following sequence5′-N¹-GTCATAGA-N²-3′ (Seq. ID No. 12) or 5′-N³-ACGCGTCC-N⁴-3′ (Seq. IDNo. 98) or 5′-N¹¹-TGTTTAGG-N¹²-3′ (Seq. ID No. 10) or5′-N⁵-TTTGGTAG-N⁶-3′ (Seq. ID No. 11) or 5′-N⁷-AATGGACC-N⁸-3′ (Seq. IDNo. 100) or 5′-N⁹-ATTAATAA-N¹⁰-3′ (Seq. ID No. 101), wherein theresidues N¹ to N¹² have the meanings especially the further limitedmeanings as disclosed herein and salts and optical isomers of saidantisense-oligonucleotide.

Moreover, the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the antisense-oligonucleotide is represented by thefollowing sequence 5′-N¹-GTCATAGA-N²-3′ (Seq. ID No. 12), wherein N¹represents: CATGGCAGACCCCGCTGCTC-, ATGGCAGACCCCGCTGCTC-,TGGCAGACCCCGCTGCTC-, GGCAGACCCCGCTGCTC-, GCAGACCCCGCTGCTC-,CAGACCCCGCTGCTC-, AGACCCCGCTGCTC-, GACCCCGCTGCTC-, ACCCCGCTGCTC-,CCCCGCTGCTC-, CCCGCTGCTC-, CCGCTGCTC-, CGCTGCTC-, GCTGCTC-, CTGCTC-,TGCTC-, GCTC-, CTC-, TC-, or C-;

N²

represents: -C, -CC, -CCG, -CCGA, -CCGAG, -CCGAGC, -CCGAGCC, -CCGAGCCC,-CCGAGCCCC, -CCGAGCCCCC, -CCGAGCCCCCA, -CCGAGCCCCCAG, -CCGAGCCCCCAGC,-CCGAGCCCCCAGCG, -CCGAGCCCCCAGCGC, -CCGAGCCCCCAGCGCA,-CCGAGCCCCCAGCGCAG, -CCGAGCCCCCAGCGCAGC, -CCGAGCCCCCAGCGCAGCG, orCCGAGCCCCCAGCGCAGCGG;and salts and optical isomers of the antisense-oligonucleotide.

The antisense-oligonucleotides of formula 51 (Seq. ID No. 12) preferablycomprise 2 to 10 LNA units, more preferably 3 to 9 LNA units and stillmore preferably 4 to 8 LNA units and also preferably at least 6 non-LNAunits, more preferably at least 7 non-LNA units and most preferably atleast 8 non-LNA units. The non-LNA units are preferably DNA units. TheLNA units are preferably positioned at the 3′ terminal end (also named3′ terminus) and the 5′ terminal end (also named 5′ terminus).Preferably at least one and more preferably at least two LNA units arepresent at the 3′ terminal end and/or at the 5′ terminal end.

Thus, preferred are antisense-oligonucleotides of the present inventiondesigned as GAPmers which contain 2 to 10 LNA units and which especiallycontain 1 to 5 LNA units at the 5′ terminal end and 1 to 5 LNA units atthe 3′ terminal end of the antisense-oligonucleotide and between the LNAunits at least 7 and more preferably at least 8 DNA units. Morepreferably the antisense-oligonucleotides comprise 2 to 4 LNA units atthe 5′ terminal end and 2 to 4 LNA units at the 3′ terminal end andstill more preferred comprise 3 to 4 LNA units at the 5′ terminal endand 3 to 4 LNA units at the 3′ terminal end and contain preferably atleast 7 non-LNA units and most preferably at least 8 non-LNA units suchas DNA units in between both LNA segments.

Moreover, the antisense-oligonucleotides may contain common nucleobasessuch as adenine, guanine, cytosine, thymine and uracil as well as commonderivatives thereof such as 5-methylcytosine or 2-aminoadenine. Theantisense-oligonucleotides of the present invention may also containmodified internucleotide bridges such as phosphorothioate orphosphorodithioate instead of phosphate bridges. Such modifications maybe present only in the LNA segments or only in the non-LNA segment ofthe antisense-oligonucleotide. As LNA units especially the residues b¹to b⁹ as disclosed herein are preferred.

Thus, preferred are antisense-oligonucleotides of the formula (51):

(Seq. ID No. 12) 5′-N¹-GTCATAGA-N²-3′whereinN¹ represents: CATGGCAGACCCCGCTGCTC-, ATGGCAGACCCCGCTGCTC-,TGGCAGACCCCGCTGCTC-, GGCAGACCCCGCTGCTC-, GCAGACCCCGCTGCTC-,CAGACCCCGCTGCTC-, AGACCCCGCTGCTC-, GACCCCGCTGCTC-, ACCCCGCTGCTC-,CCCCGCTGCTC-, CCCGCTGCTC-, CCGCTGCTC-, CGCTGCTC-, GCTGCTC-, CTGCTC-,TGCTC-, GCTC-, CTC-, TC-, or C-;andN² is selected from: -C, -CC, -CCG, -CCGA, -CCGAG, -CCGAGC, -CCGAGCC,-CCGAGCCC, -CCGAGCCCC, -CCGAGCCCCC, -CCGAGCCCCCA, -CCGAGCCCCCAG,-CCGAGCCCCCAGC, -CCGAGCCCCCAGCG, -CCGAGCCCCCAGCGC, -CCGAGCCCCCAGCGCA,-CCGAGCCCCCAGCGCAG, -CCGAGCCCCCAGCGCAGC, -CCGAGCCCCCAGCGCAGCG, or-CCGAGCCCCCAGCGCAGCGG.

Preferably the antisense-oligonucleotide of general formula (S1) hasbetween 10 and 28 nucleotides and at least one LNA nucleotide at the 3′terminus and at least one LNA nucleotide at the 5′ terminus. As LNAnucleotides (LNA units) especially these disclosed in the chapter“Locked Nucleic Acids (LNA®)” and preferably in the chapter “PreferredLNAs” are suitable and as internucleotides bridges especially thesedisclosed in the chapter “Internucleotide Linkages (IL)” are suitable.

More preferably the antisense-oligonucleotide of general formula (S1)has between 11 and 24 nucleotides and at least two LNA nucleotides atthe 3′ terminus and at least two LNA nucleotides at the 5′ terminus.

Still more preferably the antisense-oligonucleotide of general formula(S1) has between 12 and 20, more preferably between 13 and 19 and stillmore preferable between 14 and 18 nucleotides and between 2 and 5,preferably 3 and 5 and more preferably between 3 and 4 LNA units at the3′ terminal end and between 2 and 5, preferably 3 and 5 and morepreferably between 3 and 4 LNA units at the 5′ terminal end. Preferablythe antisense-oligonucleotides are GAPmers of the form LNA segment A-DNAsegment-LNA segment B. Preferably the antisense-oligonucleotides containat least 6, more preferably at least 7 and most preferably at least 8non-LNA units such as DNA units in between the two LNA segments.Suitable nucleobases for the non-LNA units and the LNA units aredisclosed in the chapter “Nucleobases”.

Further preferred are antisense-oligonucleotides of the formula (S1):

5′-N¹-GTCATAGA-N²-3′whereinN¹ represents: TGGCAGACCCCGCTGCTC-, GGCAGACCCCGCTGCTC-,GCAGACCCCGCTGCTC-, CAGACCCCGCTGCTC-, AGACCCCGCTGCTC-, GACCCCGCTGCTC-,ACCCCGCTGCTC-, CCCCGCTGCTC-, CCCGCTGCTC-, CCGCTGCTC-, CGCTGCTC-,GCTGCTC-, CTGCTC-, TGCTC-, GCTC-, CTC-, TC-, or C-;andN² is selected from: -C, -CC, -CCG, -CCGA, -CCGAG, -CCGAGC, -CCGAGCC,-CCGAGCCC, -CCGAGCCCC, -CCGAGCCCCC, -CCGAGCCCCCA, -CCGAGCCCCCAG,-CCGAGCCCCCAGC, -CCGAGCCCCCAGCG, -CCGAGCCCCCAGCGC, -CCGAGCCCCCAGCGCA,-CCGAGCCCCCAGCGCAG, or -CCGAGCCCCCAGCGCAGC.

Also preferred are antisense-oligonucleotides of the formula (S1):

5′-N¹-GTCATAGA-N²-3′whereinN¹ represents: GACCCCGCTGCTC-, ACCCCGCTGCTC-, CCCCGCTGCTC-, CCCGCTGCTC-,CCGCTGCTC-, CGCTGCTC-, GCTGCTC-, CTGCTC-, TGCTC-, GCTC-, CTC-, TC-, orC-;andN² is selected from: -C, -CC, -CCG, -CCGA, -CCGAG, -CCGAGC, -CCGAGCC,-CCGAGCCC, -CCGAGCCCC, -CCGAGCCCCC, -CCGAGCCCCCA, -CCGAGCCCCCAG, or-CCGAGCCCCCAGC.

Also preferred are antisense-oligonucleotides of the formula (S1):

5′-N¹-GTCATAGA-N²-3′whereinN¹ represents: CGCTGCTC-, GCTGCTC-, CTGCTC-, TGCTC-, GCTC-, CTC-, TC-,or C-;andN² is selected from: -C, -CC, -CCG, -CCGA, -CCGAG, -CCGAGC, -CCGAGCC, or-CCGAGCCC.

Preferably, the present invention is directed toantisense-oligonucleotide(s) consisting of 12 to 24 nucleotides and atleast three of the 12 to 24 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the antisense-oligonucleotide is represented by thefollowing sequence 5′-N¹A-CGTCATAGAC-N²A-3′ (Seq. ID No. 69), whereinN^(1A) represents: CATGGCAGACCCCGCTGCT- (Seq. ID No. 657),ATGGCAGACCCCGCTGCT- (Seq. ID No. 658), TGGCAGACCCCGCTGCT- (Seq. ID No.659), GGCAGACCCCGCTGCT- (Seq. ID No. 660), GCAGACCCCGCTGCT- (Seq. ID No.661), CAGACCCCGCTGCT- (Seq. ID No. 662), AGACCCCGCTGCT- (Seq. ID No.663), GACCCCGCTGCT- (Seq. ID No. 664), ACCCCGCTGCT- (Seq. ID No. 665),CCCCGCTGCT- (Seq. ID No. 666), CCCGCTGCT-, CCGCTGCT-, CGCTGCT-, GCTGCT-,CTGCT-, TGCT-, GCT-, CT-, or T-;

N^(2A)

represents: -C, -CG, -CGA, -CGAG, -CGAGC, -CGAGCC, -CGAGCCC, -CGAGCCCC,-CGAGCCCCC, -CGAGCCCCCA (Seq. ID No. 667), -CGAGCCCCCAG (Seq. ID No.668), -CGAGCCCCCAGC (Seq. ID No. 669), -CGAGCCCCCAGCG (Seq. ID No. 670),-CGAGCCCCCAGCGC (Seq. ID No. 671), -CGAGCCCCCAGCGCA (Seq. ID No. 672),-CGAGCCCCCAGCGCAG (Seq. ID No. 673), -CGAGCCCCCAGCGCAGC (Seq. ID No.674), -CGAGCCCCCAGCGCAGCG (Seq. ID No. 675), or CGAGCCCCCAGCGCAGCGG(Seq. ID No. 676);and salts and optical isomers of the antisense-oligonucleotide.

Preferably N^(1A) represents: TGGCAGACCCCGCTGCT-, GGCAGACCCCGCTGCT-,GCAGACCCCGCTGCT-, CAGACCCCGCTGCT-, AGACCCCGCTGCT-, GACCCCGCTGCT-,ACCCCGCTGCT-, CCCCGCTGCT-, CCCGCTGCT-, CCGCTGCT-, CGCTGCT-, GCTGCT-,CTGCT-, TGCT-, GCT-, CT-, or T-;

and

N^(2A)

represents: -C, -CG, -CGA, -CGAG, -CGAGC, -CGAGCC, -CGAGCCC, -CGAGCCCC,-CGAGCCCCC, -CGAGCCCCCA, -CGAGCCCCCAG, -CGAGCCCCCAGC, -CGAGCCCCCAGCG,-CGAGCCCCCAGCGC, -CGAGCCCCCAGCGCA, -CGAGCCCCC AGCGCAG, or-CGAGCCCCCAGCGCAGC.

More preferably N^(1A) represents: GACCCCGCTGCT-, ACCCCGCTGCT-,CCCCGCTGCT-, CCCGCTGCT-, CCGCTGCT-, CGCTGCT-, GCTGCT-, CTGCT-, TGCT-,GCT-, CT-, or T-; and

N^(2A)

represents: -C, -CG, -CGA, -CGAG, -CGAGC, -CGAGCC, -CGAGCCC, -CGAGCCCC,-CGAGCCCCC, -CGAGCCCCCA, -CGAGCCCCCAG, or -CGAGCCCCCAGC.

Still more preferably N^(1A) represents: CGCTGCT-, GCTGCT-, CTGCT-,TGCT-, GCT-, CT-, or T-; and

N^(2A) represents: -C, -CG, -CGA, -CGAG, -CGAGC, -CGAGCC, or -CGAGCCC.

Preferably the antisense-oligonucleotide of general formula (S1A/Seq. IDNo. 69) has between 12 and 24 nucleotides and at least one LNAnucleotide at the 3′ terminus and at least one LNA nucleotide at the 5′terminus. As LNA nucleotides (LNA units) especially these disclosed inthe chapter “Locked Nucleic Acids (LNA®)” and preferably in the chapter“Preferred LNAs” are suitable and as internucleotides bridges especiallythese disclosed in the chapter “Internucleotide Linkages (IL)” aresuitable.

More preferably the antisense-oligonucleotide of general formula (S1A)has between 12 and 22 nucleotides and at least two LNA nucleotides atthe 3′ terminus and at least two LNA nucleotides at the 5′ terminus.

Still more preferably the antisense-oligonucleotide of general formula(S1A) has between 12 and 20, more preferably between 13 and 19 and stillmore preferable between 14 and 18 nucleotides and between 2 and 5,preferably 3 and 5 and more preferably between 3 and 4 LNA units at the3′ terminal end and between 2 and 5, preferably 3 and 5 and morepreferably between 3 and 4 LNA units at the 5′ terminal end. Preferablythe antisense-oligonucleotides are GAPmers of the form LNA segment A-DNAsegment-LNA segment B. Preferably the antisense-oligonucleotides containat least 6, more preferably at least 7 and most preferably at least 8non-LNA units such as DNA units in between the two LNA segments.Suitable nucleobases for the non-LNA units and the LNA units aredisclosed in the chapter “Nucleobases”.

Moreover, the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the antisense-oligonucleotide is represented by thefollowing sequence 5′-N³-ACGCGTCC-N⁴-3′ (Seq. ID No. 98), wherein

N³ represents: GGTGGGATCGTGCTGGCGAT-, GTGGGATCGTGCTGGCGAT-,TGGGATCGTGCTGGCGAT-, GGGATCGTGCTGGCGAT-, GGATCGTGCTGGCGAT-,GATCGTGCTGGCGAT-, ATCGTGCTGGCGAT-, TCGTGCTGGCGAT-, CGTGCTGGCGAT-,GTGCTGGCGAT-, TGCTGGCGAT-, GCTGGCGAT-, CTGGCGAT-, TGGCGAT-, GGCGAT-,GCGAT-, CGAT-, GAT-, AT-, or T-;

N⁴

represents: -ACAGGACGATGTGCAGCGGC, -ACAGGACGATGTGCAGCGG,-ACAGGACGATGTGCAGCG, -ACAGGACGATGTGCAGC, -ACAGGACGATGTGCAG,-ACAGGACGATGTGCA, -ACAGGACGATGTGC, -ACAGGACGATGTG, -ACAGGACGATGT,-ACAGGACGATG, -ACAGGACGAT, -ACAGGACGA, -ACAGGACG, -ACAGGAC, -ACAGGA,-ACAGG, -ACAG, -ACA, -AC, or -A;and salts and optical isomers of the antisense-oligonucleotide.

The antisense-oligonucleotides of formula S2 (Seq. ID No. 98) preferablycomprise 2 to 10 LNA units, more preferably 3 to 9 LNA units and stillmore preferably 4 to 8 LNA units and also preferably at least 6 non-LNAunits, more preferably at least 7 non-LNA units and most preferably atleast 8 non-LNA units. The non-LNA units are preferably DNA units. TheLNA units are preferably positioned at the 3′ terminal end (also named3′ terminus) and the 5′ terminal end (also named 5′ terminus).Preferably at least one and more preferably at least two LNA units arepresent at the 3′ terminal end and/or at the 5′ terminal end.

Thus, preferred are antisense-oligonucleotides of the present inventiondesigned as GAPmers which contain 2 to 10 LNA units and which especiallycontain 1 to 5 LNA units at the 5′ terminal end and 1 to 5 LNA units atthe 3′ terminal end of the antisense-oligonucleotide and between the LNAunits at least 7 and more preferably at least 8 DNA units. Morepreferably the antisense-oligonucleotides comprise 2 to 4 LNA units atthe 5′ terminal end and 2 to 4 LNA units at the 3′ terminal end andstill more preferred comprise 3 to 4 LNA units at the 5′ terminal endand 3 to 4 LNA units at the 3′ terminal end and contain preferably atleast 7 non-LNA units and most preferably at least 8 non-LNA units suchas DNA units in between both LNA segments.

Moreover the antisense-oligonucleotides may contain common nucleobasessuch as adenine, guanine, cytosine, thymine and uracil as well as commonderivatives thereof such as 5-methylcytosine or 2-aminoadenine. Theantisense-oligonucleotides of the present invention may also containmodified internucleotide bridges such as phosphorothioate orphosphorodithioate instead of phosphate bridges. Such modifications maybe present only in the LNA segments or only in the non-LNA segment ofthe antisense-oligonucleotide. As LNA units especially the residues b¹to b⁹ as disclosed herein are preferred.

Thus, preferred are antisense-oligonucleotides of the formula (S2):

(Seq. ID No. 98) 5′-N³-ACGCGTCC-N⁴-3′whereinN³ represents: GGTGGGATCGTGCTGGCGAT-, GTGGGATCGTGCTGGCGAT-,TGGGATCGTGCTGGCGAT-, GGGATCGTGCTGGCGAT-, GGATCGTGCTGGCGAT-,GATCGTGCTGGCGAT-, ATCGTGCTGGCGAT-, TCGTGCTGGCGAT-, CGTGCTGGCGAT-,GTGCTGGCGAT-, TGCTGGCGAT-, GCTGGCGAT-, CTGGCGAT-, TGGCGAT-, GGCGAT-,GCGAT-, CGAT-, GAT-, AT-, or T-;and

N⁴

represents: -ACAGGACGATGTGCAGCGGC, -ACAGGACGATGTGCAGCGG,-ACAGGACGATGTGCAGCG, -ACAGGACGATGTGCAGC, -ACAGGACGATGTGCAG,-ACAGGACGATGTGCA, -ACAGGACGATGTGC, -ACAGGACGATGTG, -ACAGGACGATGT,-ACAGGACGATG, -ACAGGACGAT, -ACAGGACGA, -ACAGGACG, -ACAGGAC, -ACAGGA,-ACAGG, -ACAG, -ACA, -AC, or -A.

Preferably the antisense-oligonucleotide of general formula (S2) hasbetween 10 and 28 nucleotides and at least one LNA nucleotide at the 3′terminus and at least one LNA nucleotide at the 5′ terminus. As LNAnucleotides (LNA units) especially these disclosed in the chapter“Locked Nucleic Acids (LNA®)” and preferably in the chapter “PreferredLNAs” are suitable and as internucleotides bridges especially thesedisclosed in the chapter “Internucleotide Linkages (IL)” are suitable.

More preferably the antisense-oligonucleotide of general formula (S2)has between 11 and 24 nucleotides and at least two LNA nucleotides atthe 3′ terminus and at least two LNA nucleotides at the 5′ terminus.

Still more preferably the antisense-oligonucleotide of general formula(S2) has between 12 and 20, more preferably between 13 and 19 and stillmore preferable between 14 and 18 nucleotides and between 2 and 5,preferably 3 and 5 and more preferably between 3 and 4 LNA units at the3′ terminal end and between 2 and 5, preferably 3 and 5 and morepreferably between 3 and 4 LNA units at the 5′ terminal end. Preferablythe antisense-oligonucleotides are GAPmers of the form LNA segment A-DNAsegment-LNA segment B. Preferably the antisense-oligonucleotides containat least 6, more preferably at least 7 and most preferably at least 8non-LNA units such as DNA units in between the two LNA segments.Suitable nucleobases for the non-LNA units and the LNA units aredisclosed in the chapter “Nucleobases”.

Further preferred are antisense-oligonucleotides of the formula (S2):

5′-N³-ACGCGTCC-N⁴-3′whereinN³ represents: TGGGATCGTGCTGGCGAT-, GGGATCGTGCTGGCGAT-,GGATCGTGCTGGCGAT-, GATCGTGCTGGCGAT-, ATCGTGCTGGCGAT-, TCGTGCTGGCGAT-,CGTGCTGGCGAT-, GTGCTGGCGAT-, TGCTGGCGAT-, GCTGGCGAT-, CTGGCGAT-,TGGCGAT-, GGCGAT-, GCGAT-, CGAT-, GAT-, AT-, or T-;and

N⁴

represents: -ACAGGACGATGTGCAGCG, -ACAGGACGATGTGCAGC, -ACAGGACGATGTGCAG,-ACAGGACGATGTGCA, -ACAGGACGATGTGC, -ACAGGACGATGTG, -ACAGGACGATGT,-ACAGGACGATG, -ACAGGACGAT, -ACAGGACGA, -ACAGGACG, -ACAGGAC, -ACAGGA,-ACAGG, -ACAG, -ACA, -AC, or -A.

Also preferred are antisense-oligonucleotides of the formula (S2):

5′-N³-ACGCGTCC-N⁴-3′whereinN³ represents: TCGTGCTGGCGAT-, CGTGCTGGCGAT-, GTGCTGGCGAT-, TGCTGGCGAT-,GCTGGCGAT-, CTGGCGAT-, TGGCGAT-, GGCGAT-, GCGAT-, CGAT-, GAT-, AT-, orT-;and

N⁴

represents: -ACAGGACGATGTG, -ACAGGACGATGT, -ACAGGACGATG, -ACAGGACGAT,-ACAGGACGA, -ACAGGACG, -ACAGGAC, -ACAGGA, -ACAGG, -ACAG, -ACA, -AC, or-A.

Also preferred are antisense-oligonucleotides of the formula (S2):

5′-N³-ACGCGTCC-N⁴-3′whereinN³ represents: CTGGCGAT-, TGGCGAT-, GGCGAT-, GCGAT-, CGAT-, GAT-, AT-,or T-;andN⁴ represents: -ACAGGACG, -ACAGGAC, -ACAGGA, -ACAGG, -ACAG, -ACA, -AC,or -A.

Preferably, the present invention is directed toantisense-oligonucleotide(s) consisting of 12 to 24 nucleotides and atleast three of the 12 to 24 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the antisense-oligonucleotide is represented by thefollowing sequence 5′-N³A-TACGCGTCCA-N⁴A-3′ (Seq. ID No. 70), wherein

N^(3A) represents: GGTGGGATCGTGCTGGCGA- (Seq. ID No. 677),GTGGGATCGTGCTGGCGA- (Seq. ID No. 678), TGGGATCGTGCTGGCGA- (Seq. ID No.679), GGGATCGTGCTGGCGA- (Seq. ID No. 680), GGATCGTGCTGGCGA- (Seq. ID No.681), GATCGTGCTGGCGA- (Seq. ID No. 682), ATCGTGCTGGCGA- (Seq. ID No.683), TCGTGCTGGCGA- (Seq. ID No. 684), CGTGCTGGCGA- (Seq. ID No. 685),GTGCTGGCGA- (Seq. ID No. 686), TGCTGGCGA-, GCTGGCGA-, CTGGCGA-, TGGCGA-,GGCGA-, GCGA-, CGA-, GA-, or A-;N^(4A) represents: -CAGGACGATGTGCAGCGGC (Seq. ID No. 687),-CAGGACGATGTGCAGCGG (Seq. ID No. 688), -CAGGACGATGTGCAGCG (Seq. ID No.689), -CAGGACGATGTGCAGC (Seq. ID No. 690), -CAGGACGATGTGCAG (Seq. ID No.691), -CAGGACGATGTGCA (Seq. ID No. 692), -CAGGACGATGTGC (Seq. ID No.693), -CAGGACGATGTG (Seq. ID No. 694), -CAGGACGATGT (Seq. ID No. 695),-CAGGACGATG (Seq. ID No. 696), -CAGGACGAT, -CAGGACGA, -CAGGACG, -CAGGAC,-CAGGA, -CAGG, -CAG, -CA, or -C;and salts and optical isomers of the antisense-oligonucleotide.

Preferably N^(3A) represents: TGGGATCGTGCTGGCGA-, GGGATCGTGCTGGCGA-,GGATCGTGCTGGCGA-, GATCGTGCTGGCGA-, ATCGTGCTGGCGA-, TCGTGCTGGCGA-,CGTGCTGGCGA-, GTGCTGGCGA-, TGCTGGCGA-, GCTGGCGA-, CTGGCGA-, TGGCGA-,GGCGA-, GCGA-, CGA-, GA-, or A-;

and

N^(4A)

represents: -CAGGACGATGTGCAGCG, -CAGGACGATGTGCAGC, -CAGGACGATGTGCAG,-CAGGACGATGTGCA, -CAGGACGATGTGC, -CAGGACGATGTG, -CAGGACGATGT,-CAGGACGATG, -CAGGACGAT, -CAGGACGA, -CAGGACG, -CAGGAC, -CAGGA, -CAGG,-CAG, -CA, or -C.

More preferably N^(3A) represents: TCGTGCTGGCGA-, CGTGCTGGCGA-,GTGCTGGCGA-, TGCTGGCGA-, GCTGGCGA-, CTGGCGA-, TGGCGA-, GGCGA-, GCGA-,CGA-, GA-, or A-; and

N^(4A)

represents: -CAGGACGATGTG, -CAGGACGATGT, -CAGGACGATG, -CAGGACGAT,-CAGGACGA, -CAGGACG, -CAGGAC, -CAGGA, -CAGG, -CAG, -CA, or -C.

Still more preferably N^(3A) represents: CTGGCGA-, TGGCGA-, GGCGA-,GCGA-, CGA-, GA-, or A-; and

N^(4A) represents: -CAGGACG, -CAGGAC, -CAGGA, -CAGG, -CAG, -CA, or -C.

Preferably the antisense-oligonucleotide of general formula (S2A/Seq. IDNo. 70) has between 12 and 24 nucleotides and at least one LNAnucleotide at the 3′ terminus and at least one LNA nucleotide at the 5′terminus. As LNA nucleotides (LNA units) especially these disclosed inthe chapter “Locked Nucleic Acids (LNA®)” and preferably in the chapter“Preferred LNAs” are suitable and as internucleotides bridges especiallythese disclosed in the chapter “Internucleotide Linkages (IL)” aresuitable.

More preferably the antisense-oligonucleotide of general formula (S2A)has between 12 and 22 nucleotides and at least two LNA nucleotides atthe 3′ terminus and at least two LNA nucleotides at the 5′ terminus.

Still more preferably the antisense-oligonucleotide of general formula(S2A) has between 12 and 20, more preferably between 13 and 19 and stillmore preferable between 14 and 18 nucleotides and between 2 and 5,preferably 3 and 5 and more preferably between 3 and 4 LNA units at the3′ terminal end and between 2 and 5, preferably 3 and 5 and morepreferably between 3 and 4 LNA units at the 5′ terminal end. Preferablythe antisense-oligonucleotides are GAPmers of the form LNA segment A-DNAsegment-LNA segment B. Preferably the antisense-oligonucleotides containat least 6, more preferably at least 7 and most preferably at least 8non-LNA units such as DNA units in between the two LNA segments.Suitable nucleobases for the non-LNA units and the LNA units aredisclosed in the chapter “Nucleobases”.

Moreover, the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the antisense-oligonucleotide is represented by thefollowing sequence 5′-N¹¹-TGTTTAGG-N¹²-3′ (Seq. ID No. 10), wherein N¹¹represents: TGCCCCAGAAGAGCTATTTGGTAG-, GCCCCAGAAGAGCTATTTGGTAG-,CCCCAGAAGAGCTATTTGGTAG-, CCCAGAAGAGCTATTTGGTAG-, CCAGAAGAGCTATTTGGTAG-,CAGAAGAGCTATTTGGTAG-, AGAAGAGCTATTTGGTAG-, GAAGAGCTATTTGGTAG-,AAGAGCTATTTGGTAG-, AGAGCTATTTGGTAG-, GAGCTATTTGGTAG-, AGCTATTTGGTAG-,GCTATTTGGTAG-, CTATTTGGTAG-, TATTTGGTAG-, ATTTGGTAG-, TTTGGTAG-,TTGGTAG-, TGGTAG-, GGTAG-, GTAG-, TAG-, AG- or G-,

N¹² represents: -GAGCCGTCTTCAGGAATCTTCTCC, -GAGCCGTCTTCAGGAATCTTCTC,-GAGCCGTCTTCAGGAATCTTCT, -GAGCCGTCTTCAGGAATCTTC, -GAGCCGTCTTCAGGAATCTT,-GAGCCGTCTTCAGGAATCT, -GAGCCGTCTTCAGGAATC, -GAGCCGTCTTCAGGAAT,-GAGCCGTCTTCAGGAA, -GAGCCGTCTTCAGGA, -GAGCCGTCTTCAGG, -GAGCCGTCTTCAG,-GAGCCGTCTTCA, -GAGCCGTCTTC, -GAGCCGTCTT, -GAGCCGTCT, -GAGCCGTC,-GAGCCGT, -GAGCCG, -GAGCC, -GAGC, -GAG, -GA, or G;and salts and optical isomers of the antisense-oligonucleotide.

The antisense-oligonucleotides of formula S3 (Seq. ID No. 10) preferablycomprise 2 to 10 LNA units, more preferably 3 to 9 LNA units and stillmore preferably 4 to 8 LNA units and also preferably at least 6 non-LNAunits, more preferably at least 7 non-LNA units and most preferably atleast 8 non-LNA units. The non-LNA units are preferably DNA units. TheLNA units are preferably positioned at the 3′ terminal end (also named3′ terminus) and the 5′ terminal end (also named 5′ terminus).Preferably at least one and more preferably at least two LNA units arepresent at the 3′ terminal end and/or at the 5′ terminal end.

Thus, preferred are antisense-oligonucleotides of the present inventiondesigned as GAPmers which contain 2 to 10 LNA units and which especiallycontain 1 to 5 LNA units at the 5′ terminal end and 1 to 5 LNA units atthe 3′ terminal end of the antisense-oligonucleotide and between the LNAunits at least 7 and more preferably at least 8 DNA units. Morepreferably the antisense-oligonucleotides comprise 2 to 4 LNA units atthe 5′ terminal end and 2 to 4 LNA units at the 3′ terminal end andstill more preferred comprise 3 to 4 LNA units at the 5′ terminal endand 3 to 4 LNA units at the 3′ terminal end and contain preferably atleast 7 non-LNA units and most preferably at least 8 non-LNA units suchas DNA units in between both LNA segments.

Moreover the antisense-oligonucleotides may contain common nucleobasessuch as adenine, guanine, cytosine, thymine and uracil as well as commonderivatives thereof such as 5-methylcytosine or 2-aminoadenine. Theantisense-oligonucleotides of the present invention may also containmodified internucleotide bridges such as phosphorothioate orphosphorodithioate instead of phosphate bridges. Such modifications maybe present only in the LNA segments or only in the non-LNA segment ofthe antisense-oligonucleotide. As LNA units especially the residues b¹to b⁹ as disclosed herein are preferred.

Thus, preferred are antisense-oligonucleotides of the formula (S3):

(Seq. ID No. 10) 5′-N¹¹-TGTTTAGG-N¹²-3′whereinN¹¹ represents: TGCCCCAGAAGAGCTATTTGGTAG-, GCCCCAGAAGAGCTATTTGGTAG-,CCCCAGAAGAGCTATTTGGTAG-, CCCAGAAGAGCTATTTGGTAG-, CCAGAAGAGCTATTTGGTAG-,CAGAAGAGCTATTTGGTAG-, AGAAGAGCTATTTGGTAG-, GAAGAGCTATTTGGTAG-,AAGAGCTATTTGGTAG-, AGAGCTATTTGGTAG-, GAGCTATTTGGTAG-, AGCTATTTGGTAG-,GCTATTTGGTAG-, CTATTTGGTAG-, TATTTGGTAG-, ATTTGGTAG-, TTTGGTAG-,TTGGTAG-, TGGTAG-, GGTAG-, GTAG-, TAG-, AG- or G-,andN¹² represents: -GAGCCGTCTTCAGGAATCTTCTCC, -GAGCCGTCTTCAGGAATCTTCTC,-GAGCCGTCTTCAGGAATCTTCT, -GAGCCGTCTTCAGGAATCTTC, -GAGCCGTCTTCAGGAATCTT,-GAGCCGTCTTCAGGAATCT, -GAGCCGTCTTCAGGAATC, -GAGCCGTCTTCAGGAAT,-GAGCCGTCTTCAGGAA, -GAGCCGTCTTCAGGA, -GAGCCGTCTTCAGG, -GAGCCGTCTTCAG,-GAGCCGTCTTCA, -GAGCCGTCTTC, -GAGCCGTCTT, -GAGCCGTCT, -GAGCCGTC,-GAGCCGT, -GAGCCG, -GAGCC, -GAGC, -GAG, -GA, or G.

Preferably the antisense-oligonucleotide of general formula (S3) hasbetween 10 and 28 nucleotides and at least one LNA nucleotide at the 3′terminus and at least one LNA nucleotide at the 5′ terminus. As LNAnucleotides (LNA units) especially these disclosed in the chapter“Locked Nucleic Acids (LNA®)” and preferably in the chapter “PreferredLNAs” are suitable and as internucleotides bridges especially thesedisclosed in the chapter “Internucleotide Linkages (IL)” are suitable.

More preferably the antisense-oligonucleotide of general formula (S3)has between 11 and 24 nucleotides and at least two LNA nucleotides atthe 3′ terminus and at least two LNA nucleotides at the 5′ terminus.

Still more preferably the antisense-oligonucleotide of general formula(S3) has between 12 and 20, more preferably between 13 and 19 and stillmore preferable between 14 and 18 nucleotides and between 2 and 5,preferably 3 and 5 and more preferably between 3 and 4 LNA units at the3′ terminal end and between 2 and 5, preferably 3 and 5 and morepreferably between 3 and 4 LNA units at the 5′ terminal end. Preferablythe antisense-oligonucleotides are GAPmers of the form LNA segment A-DNAsegment-LNA segment B. Preferably the antisense-oligonucleotides containat least 6, more preferably at least 7 and most preferably at least 8non-LNA units such as DNA units in between the two LNA segments.Suitable nucleobases for the non-LNA units and the LNA units aredisclosed in the chapter “Nucleobases”.

Further preferred are antisense-oligonucleotides of the formula (S3):

5′-N¹¹-TGTTTAGG-N¹²-3′whereinN¹¹ represents: AGAAGAGCTATTTGGTAG-, GAAGAGCTATTTGGTAG-,AAGAGCTATTTGGTAG-, AGAGCTATTTGGTAG-, GAGCTATTTGGTAG-, AGCTATTTGGTAG-,GCTATTTGGTAG-, CTATTTGGTAG-, TATTTGGTAG-, ATTTGGTAG-, TTTGGTAG-,TTGGTAG-, TGGTAG-, GGTAG-, GTAG-, TAG-, AG- or G-;andN¹² represents: -GAGCCGTCTTCAGGAATC, -GAGCCGTCTTCAGGAAT,-GAGCCGTCTTCAGGAA, -GAGCCGTCTTCAGGA, -GAGCCGTCTTCAGG, -GAGCCGTCTTCAG,-GAGCCGTCTTCA, -GAGCCGTCTTC, -GAGCCGTCTT, -GAGCCGTCT, -GAGCCGTC,-GAGCCGT, -GAGCCG, -GAGCC, -GAGC, -GAG, -GA, or G.

Also preferred are antisense-oligonucleotides of the formula (S3):

5′-N¹¹-TGTTTAGG-N¹²-3′whereinN¹¹ represents: AGCTATTTGGTAG-, GCTATTTGGTAG-, CTATTTGGTAG-,TATTTGGTAG-, ATTTGGTAG-, TTTGGTAG-, TTGGTAG-, TGGTAG-, GGTAG-, GTAG-,TAG-, AG- or G-;andN¹² represents: -GAGCCGTCTTCAG, -GAGCCGTCTTCA, -GAGCCGTCTTC,-GAGCCGTCTT, -GAGCCGTCT, -GAGCCGTC, -GAGCCGT, -GAGCCG, -GAGCC, -GAGC,-GAG, -GA, or G.

Also preferred are antisense-oligonucleotides of the formula (S3):

5′-N¹¹-TGTTTAGG-N¹²-3′whereinN¹¹ represents: TTTGGTAG-, TTGGTAG-, TGGTAG-, GGTAG-, GTAG-, TAG-, AG-or G-;andN¹² represents: -GAGCCGTC, -GAGCCGT, -GAGCCG, -GAGCC, -GAGC, -GAG, -GA,or G.

Preferably, the present invention is directed toantisense-oligonucleotide(s) consisting of 12 to 24 nucleotides and atleast three of the 12 to 24 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the antisense-oligonucleotide is represented by thefollowing sequence 5′-N¹¹A-GTGTTTAGGG-N¹²A-3′ (Seq. ID No. 71), wherein

N^(11A) represents: TGCCCCAGAAGAGCTATTTGGTA- (Seq. ID No. 765),GCCCCAGAAGAGCTATTTGGTA- (Seq. ID No. 766), CCCCAGAAGAGCTATTTGGTA- (Seq.ID No. 767), CCCAGAAGAGCTATTTGGTA- (Seq. ID No. 768),CCAGAAGAGCTATTTGGTA- (Seq. ID No. 769), CAGAAGAGCTATTTGGTA- (Seq. ID No.770), AGAAGAGCTATTTGGTA- (Seq. ID No. 771), GAAGAGCTATTTGGTA- (Seq. IDNo. 772), AAGAGCTATTTGGTA- (Seq. ID No. 773), AGAGCTATTTGGTA- (Seq. IDNo. 774), GAGCTATTTGGTA- (Seq. ID No. 775), AGCTATTTGGTA- (Seq. ID No.776), GCTATTTGGTA- (Seq. ID No. 777), CTATTTGGTA- (Seq. ID No. 778),TATTTGGTA-, ATTTGGTA-, TTTGGTA-, TTGGTA-, TGGTA-, GGTA-, GTA-, TA-, orA-,N^(12A) represents: -AGCCGTCTTCAGGAATCTTCTCC (Seq. ID No. 779),-AGCCGTCTTCAGGAATCTTCTC (Seq. ID No. 780), AGCCGTCTTCAGGAATCTTCT (Seq.ID No. 781), -AGCCGTCTTCAGGAATCTTC (Seq. ID No. 782),-AGCCGTCTTCAGGAATCTT (Seq. ID No. 783), -AGCCGTCTTCAGGAATCT (Seq. ID No.784), -AGCCGTCTTCAGGAATC (Seq. ID No. 785), -AGCCGTCTTCAGGAAT (Seq. IDNo. 786), -AGCCGTCTTCAGGAA (Seq. ID No. 787), -AGCCGTCTTCAGGA (Seq. IDNo. 788), -AGCCGTCTTCAGG (Seq. ID No. 789), -AGCCGTCTTCAG (Seq. ID No.790), -AGCCGTCTTCA (Seq. ID No. 791), -AGCCGTCTTC (Seq. ID No. 792),-AGCCGTCTT, -AGCCGTCT, -AGCCGTC, -AGCCGT, -AGCCG, -AGCC, -AGC, -AG, or-A;and salts and optical isomers of the antisense-oligonucleotide.

Preferably N^(11A) represents: AGAAGAGCTATTTGGTA-, GAAGAGCTATTTGGTA-,AAGAGCTATTTGGTA-, AGAGCTATTTGGTA-, GAGCTATTTGGTA-, AGCTATTTGGTA-,GCTATTTGGTA-, CTATTTGGTA-, TATTTGGTA-, ATTTGGTA-, TTTGGTA-, TTGGTA-,TGGTA-, GGTA-, GTA-, TA-, or A-; and

N^(12A) represents: -AGCCGTCTTCAGGAATC, -AGCCGTCTTCAGGAAT,-AGCCGTCTTCAGGAA, -AGCCGTCTTCAGGA, -AGCCGTCTTCAGG, -AGCCGTCTTCAG,-AGCCGTCTTCA, -AGCCGTCTTC, -AGCCGTCTT, -AGCCGTCT, -AGCCGTC, -AGCCGT,-AGCCG, -AGCC, -AGC, -AG, or -A.

More preferably N^(11A) represents: AGCTATTTGGTA-, GCTATTTGGTA-,CTATTTGGTA-, TATTTGGTA-, ATTTGGTA-, TTTGGTA-, TTGGTA-, TGGTA-, GGTA-,GTA-, TA-, or A-; and

N^(12A) represents: -AGCCGTCTTCAG, -AGCCGTCTTCA, -AGCCGTCTTC,-AGCCGTCTT, -AGCCGTCT, -AGCCGTC, -AGCCGT, -AGCCG, -AGCC, -AGC, -AG, or-A.

Still more preferably N^(11A) represents: TTTGGTA-, TTGGTA-, TGGTA-,GGTA-, GTA-, TA-, or A-; and

N^(12A) represents: -AGCCGTC, -AGCCGT, -AGCCG, -AGCC, -AGC, -AG, or -A.

Preferably the antisense-oligonucleotide of general formula (S3A/Seq. IDNo. 71) has between 12 and 24 nucleotides and at least one LNAnucleotide at the 3′ terminus and at least one LNA nucleotide at the 5′terminus. As LNA nucleotides (LNA units) especially these disclosed inthe chapter “Locked Nucleic Acids (LNA®)” and preferably in the chapter“Preferred LNAs” are suitable and as internucleotides bridges especiallythese disclosed in the chapter “Internucleotide Linkages (IL)” aresuitable.

More preferably the antisense-oligonucleotide of general formula (S3A)has between 12 and 22 nucleotides and at least two LNA nucleotides atthe 3′ terminus and at least two LNA nucleotides at the 5′ terminus.

Still more preferably the antisense-oligonucleotide of general formula(S3A) has between 12 and 20, more preferably between 13 and 19 and stillmore preferable between 14 and 18 nucleotides and between 2 and 5,preferably 3 and 5 and more preferably between 3 and 4 LNA units at the3′ terminal end and between 2 and 5, preferably 3 and 5 and morepreferably between 3 and 4 LNA units at the 5′ terminal end. Preferablythe antisense-oligonucleotides are GAPmers of the form LNA segment A-DNAsegment-LNA segment B. Preferably the antisense-oligonucleotides containat least 6, more preferably at least 7 and most preferably at least 8non-LNA units such as DNA units in between the two LNA segments.Suitable nucleobases for the non-LNA units and the LNA units aredisclosed in the chapter “Nucleobases”.

Moreover, the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the antisense-oligonucleotide is represented by thefollowing sequence 5′-N⁵-TTTGGTAG-N⁶-3′ (Seq. ID No. 11), wherein

N⁵ represents: GCCCAGCCTGCCCCAGAAGAGCTA-, CCCAGCCTGCCCCAGAAGAGCTA-,CCAGCCTGCCCCAGAAGAGCTA-, CAGCCTGCCCCAGAAGAGCTA-, AGCCTGCCCCAGAAGAGCTA-,GCCTGCCCCAGAAGAGCTA-, CCTGCCCCAGAAGAGCTA-, CTGCCCCAGAAGAGCTA-,TGCCCCAGAAGAGCTA-, GCCCCAGAAGAGCTA-, CCCCAGAAGAGCTA-, CCCAGAAGAGCTA-,CCAGAAGAGCTA-, CAGAAGAGCTA-, AGAAGAGCTA-, GAAGAGCTA-, AAGAGCTA-,AGAGCTA-, GAGCTA-, AGCTA-, GCTA-, CTA-, TA-, or A-;N⁶ represents: -TGTTTAGGGAGCCGTCTTCAGGAA, -TGTTTAGGGAGCCGTCTTCAGGA,-TGTTTAGGGAGCCGTCTTCAGG, -TGTTTAGGGAGCCGTCTTCAG, -TGTTTAGGGAGCCGTCTTCA,-TGTTTAGGGAGCCGTCTTC, -TGTTTAGGGAGCCGTCTT, -TGTTTAGGGAGCCGTCT,-TGTTTAGGGAGCCGTC, -TGTTTAGGGAGCCGT, -TGTTTAGGGAGCCG, -TGTTTAGGGAGCC,-TGTTTAGGGAGC, -TGTTTAGGGAG, -TGTTTAGGGA, -TGTTTAGGG, -TGTTTAGG,-TGTTTAG, -TGTTTA, -TGTTT, -TGTT, -TGT, -TG, or T;and salts and optical isomers of the antisense-oligonucleotide.

The antisense-oligonucleotides of formula S4 (Seq. ID No. 11) preferablycomprise 2 to 10 LNA units, more preferably 3 to 9 LNA units and stillmore preferably 4 to 8 LNA units and also preferably at least 6 non-LNAunits, more preferably at least 7 non-LNA units and most preferably atleast 8 non-LNA units. The non-LNA units are preferably DNA units. TheLNA units are preferably positioned at the 3′ terminal end (also named3′ terminus) and the 5′ terminal end (also named 5′ terminus).Preferably at least one and more preferably at least two LNA units arepresent at the 3′ terminal end and/or at the 5′ terminal end.

Thus, preferred are antisense-oligonucleotides of the present inventiondesigned as GAPmers which contain 2 to 10 LNA units and which especiallycontain 1 to 5 LNA units at the 5′ terminal end and 1 to 5 LNA units atthe 3′ terminal end of the antisense-oligonucleotide and between the LNAunits at least 7 and more preferably at least 8 DNA units. Morepreferably the antisense-oligonucleotides comprise 2 to 4 LNA units atthe 5′ terminal end and 2 to 4 LNA units at the 3′ terminal end andstill more preferred comprise 3 to 4 LNA units at the 5′ terminal endand 3 to 4 LNA units at the 3′ terminal end and contain preferably atleast 7 non-LNA units and most preferably at least 8 non-LNA units suchas DNA units in between both LNA segments.

Moreover the antisense-oligonucleotides may contain common nucleobasessuch as adenine, guanine, cytosine, thymine and uracil as well as commonderivatives thereof such as 5-methylcytosine or 2-aminoadenine. Theantisense-oligonucleotides of the present invention may also containmodified internucleotide bridges such as phosphorothioate orphosphorodithioate instead of phosphate bridges. Such modifications maybe present only in the LNA segments or only in the non-LNA segment ofthe antisense-oligonucleotide. As LNA units especially the residues b¹to b⁹ as disclosed herein are preferred.

Thus, preferred are antisense-oligonucleotides of the formula (S4):

(Seq. ID No. 11) 5′-N⁵-TTTGGTAG-N⁶-3′whereinN⁵ represents: GCCCAGCCTGCCCCAGAAGAGCTA-, CCCAGCCTGCCCCAGAAGAGCTA-,CCAGCCTGCCCCAGAAGAGCTA-, CAGCCTGCCCCAGAAGAGCTA-, AGCCTGCCCCAGAAGAGCTA-,GCCTGCCCCAGAAGAGCTA-, CCTGCCCCAGAAGAGCTA-, CTGCCCCAGAAGAGCTA-,TGCCCCAGAAGAGCTA-, GCCCCAGAAGAGCTA-, CCCCAGAAGAGCTA-, CCCAGAAGAGCTA-,CCAGAAGAGCTA-, CAGAAGAGCTA-, AGAAGAGCTA-, GAAGAGCTA-, AAGAGCTA-,AGAGCTA-, GAGCTA-, AGCTA-, GCTA-, CTA-, TA-, or A-;andN⁶ is selected from: -TGTTTAGGGAGCCGTCTTCAGGAA,-TGTTTAGGGAGCCGTCTTCAGGA, -TGTTTAGGGAGCCGTCTTCAGG,-TGTTTAGGGAGCCGTCTTCAG, -TGTTTAGGGAGCCGTCTTCA, -TGTTTAGGGAGCCGTCTTC,-TGTTTAGGGAGCCGTCTT, -TGTTTAGGGAGCCGTCT, -TGTTTAGGGAGCCGTC,-TGTTTAGGGAGCCGT, -TGTTTAGGGAGCCG, -TGTTTAGGGAGCC, -TGTTTAGGGAGC,-TGTTTAGGGAG, -TGTTTAGGGA, -TGTTTAGGG, -TGTTTAGG, -TGTTTAG, -TGTTTA,-TGTTT, -TGTT, -TGT, -TG, or -T.

Preferably the antisense-oligonucleotide of general formula (S4) hasbetween 10 and 28 nucleotides and at least one LNA nucleotide at the 3′terminus and at least one LNA nucleotide at the 5′ terminus. As LNAnucleotides (LNA units) especially these disclosed in the chapter“Locked Nucleic Acids (LNA®)” and preferably in the chapter “PreferredLNAs” are suitable and as internucleotides bridges especially thesedisclosed in the chapter “Internucleotide Linkages (IL)” are suitable.

More preferably the antisense-oligonucleotide of general formula (S4)has between 11 and 24 nucleotides and at least two LNA nucleotides atthe 3′ terminus and at least two LNA nucleotides at the 5′ terminus.

Still more preferably the antisense-oligonucleotide of general formula(S4) has between 12 and 20, more preferably between 13 and 19 and stillmore preferable between 14 and 18 nucleotides and between 2 and 5,preferably 3 and 5 and more preferably between 3 and 4 LNA units at the3′ terminal end and between 2 and 5, preferably 3 and 5 and morepreferably between 3 and 4 LNA units at the 5′ terminal end. Preferablythe antisense-oligonucleotides are GAPmers of the form LNA segment A-DNAsegment-LNA segment B. Preferably the antisense-oligonucleotides containat least 6, more preferably at least 7 and most preferably at least 8non-LNA units such as DNA units in between the two LNA segments.Suitable nucleobases for the non-LNA units and the LNA units aredisclosed in the chapter “Nucleobases”.

Further preferred are antisense-oligonucleotides of the formula (S4):

5′-N⁵-TTTGGTAG-N⁶-3′whereinN⁵ represents: CCTGCCCCAGAAGAGCTA-, CTGCCCCAGAAGAGCTA-,TGCCCCAGAAGAGCTA-, GCCCCAGAAGAGCTA-, CCCCAGAAGAGCTA-, CCCAGAAGAGCTA-,CCAGAAGAGCTA-, CAGAAGAGCTA-, AGAAGAGCTA-, GAAGAGCTA-, AAGAGCTA-,AGAGCTA-, GAGCTA-, AGCTA-, GCTA-, CTA-, TA-, or A-;andN⁶ is selected from: -TGTTTAGGGAGCCGTCTT, -TGTTTAGGGAGCCGTCT,-TGTTTAGGGAGCCGTC, -TGTTTAGGGAGCCGT, -TGTTTAGGGAGCCG, -TGTTTAGGGAGCC,-TGTTTAGGGAGC, -TGTTTAGGGAG, -TGTTTAGGGA, -TGTTTAGGG, -TGTTTAGG,-TGTTTAG, -TGTTTA, -TGTTT, -TGTT, -TGT, -TG, or -T.

Also preferred are antisense-oligonucleotides of the formula (S4):

5′-N⁵-TTTGGTAG-N⁶-3′whereinN⁵ represents: CCCAGAAGAGCTA-, CCAGAAGAGCTA-, CAGAAGAGCTA-, AGAAGAGCTA-,GAAGAGCTA-, AAGAGCTA-, AGAGCTA-, GAGCTA-, AGCTA-, GCTA-, CTA-, TA-, orA-;andN⁶ is selected from: -TGTTTAGGGAGCC, -TGTTTAGGGAGC, -TGTTTAGGGAG,-TGTTTAGGGA, -TGTTTAGGG, -TGTTTAGG, -TGTTTAG, -TGTTTA, -TGTTT, -TGTT,-TGT, -TG, or -T.

Also preferred are antisense-oligonucleotides of the formula (S4):

5′-N⁵-TTTGGTAG-N⁶-3′whereinN⁵ represents: AAGAGCTA-, AGAGCTA-, GAGCTA-, AGCTA-, GCTA-, CTA-, TA-,or A-;andN⁶ is selected from: -TGTTTAGG, -TGTTTAG, -TGTTTA, -TGTTT, -TGTT, -TGT,-TG, or -T.

Preferably, the present invention is directed toantisense-oligonucleotide(s) consisting of 12 to 24 nucleotides and atleast three of the 12 to 24 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the antisense-oligonucleotide is represented by thefollowing sequence 5′-N⁵A-ATTTGGTAGT-N⁶A-3′ (Seq. ID No. 72), wherein

N^(5A) represents: GCCCAGCCTGCCCCAGAAGAGCT- (Seq. ID No. 697),CCCAGCCTGCCCCAGAAGAGCT- (Seq. ID No. 698), CCAGCCTGCCCCAGAAGAGCT- (Seq.ID No. 699), CAGCCTGCCCCAGAAGAGCT- (Seq. ID No. 700),AGCCTGCCCCAGAAGAGCT- (Seq. ID No. 701), GCCTGCCCCAGAAGAGCT- (Seq. ID No.702), CCTGCCCCAGAAGAGCT- (Seq. ID No. 703), CTGCCCCAGAAGAGCT- (Seq. IDNo. 704), TGCCCCAGAAGAGCT- (Seq. ID No. 705), GCCCCAGAAGAGCT- (Seq. IDNo. 706), CCCCAGAAGAGCT- (Seq. ID No. 707), CCCAGAAGAGCT- (Seq. ID No.708), CCAGAAGAGCT- (Seq. ID No. 709), CAGAAGAGCT- (Seq. ID No. 710),AGAAGAGCT-, GAAGAGCT-, AAGAGCT-, AGAGCT-, GAGCT-, AGCT-, GCT-, CT-, orT-;N^(6A) represents: -GTTTAGGGAGCCGTCTTCAGGAA (Seq. ID No. 711),-GTTTAGGGAGCCGTCTTCAGGA (Seq. ID No. 712), GTTTAGGGAGCCGTCTTCAGG (Seq.ID No. 713), -GTTTAGGGAGCCGTCTTCAG (Seq. ID No. 714),-GTTTAGGGAGCCGTCTTCA (Seq. ID No. 715), -GTTTAGGGAGCCGTCTTC (Seq. ID No.716), -GTTTAGGGAGCCGTCTT (Seq. ID No. 717), -GTTTAGGGAGCCGTCT (Seq. IDNo. 718), -GTTTAGGGAGCCGTC (Seq. ID No. 719), -GTTTAGGGAGCCGT (Seq. IDNo. 720), -GTTTAGGGAGCCG (Seq. ID No. 721), -GTTTAGGGAGCC (Seq. ID No.722), -GTTTAGGGAGC (Seq. ID No. 723), -GTTTAGGGAG (Seq. ID No. 724),-GTTTAGGGA, -GTTTAGGG, -GTTTAGG, -GTTTAG, -GTTTA, -GTTT, -GTT, -GT, or-G;and salts and optical isomers of the antisense-oligonucleotide.

Preferably N^(5A) represents: CCTGCCCCAGAAGAGCT-, CTGCCCCAGAAGAGCT-,TGCCCCAGAAGAGCT-, GCCCCAGAAGAGCT-, CCCCAGAAGAGCT-, CCCAGAAGAGCT-,CCAGAAGAGCT-, CAGAAGAGCT-, AGAAGAGCT-, GAAGAGCT-, AAGAGCT-, AGAGCT-,GAGCT-, AGCT-, GCT-, CT-, or T-;

andN^(6A) represents: -GTTTAGGGAGCCGTCTT, -GTTTAGGGAGCCGTCT,-GTTTAGGGAGCCGTC, -GTTTAGGGAGCCGT, -GTTTAGGGAGCCG, -GTTTAGGGAGCC,-GTTTAGGGAGC, -GTTTAGGGAG, -GTTTAGGGA, -GTTTAGGG, -GTTTAGG, -GTTTAG,-GTTTA, -GTTT, -GTT, -GT, or -G.

More preferably N^(5A) represents: CCCAGAAGAGCT-, CCAGAAGAGCT-,CAGAAGAGCT-, AGAAGAGCT-, GAAGAGCT-, AAGAGCT-, AGAGCT-, GAGCT-, AGCT-,GCT-, CT-, or T-; and

N^(6A) represents: -GTTTAGGGAGCC, -GTTTAGGGAGC, -GTTTAGGGAG, -GTTTAGGGA,-GTTTAGGG, -GTTTAGG, -GTTTAG, -GTTTA, -GTTT, -GTT, -GT, or -G.

Still more preferably N^(5A) represents: AAGAGCT-, AGAGCT-, GAGCT-,AGCT-, GCT-, CT-, or T-; and

N^(6A) represents: -GTTTAGG, -GTTTAG, -GTTTA, -GTTT, -GTT, -GT, or -G.

Preferably the antisense-oligonucleotide of general formula (S4A/Seq. IDNo. 72) has between 12 and 24 nucleotides and at least one LNAnucleotide at the 3′ terminus and at least one LNA nucleotide at the 5′terminus. As LNA nucleotides (LNA units) especially these disclosed inthe chapter “Locked Nucleic Acids (LNA®)” and preferably in the chapter“Preferred LNAs” are suitable and as internucleotides bridges especiallythese disclosed in the chapter “Internucleotide Linkages (IL)” aresuitable.

More preferably the antisense-oligonucleotide of general formula (S4A)has between 12 and 22 nucleotides and at least two LNA nucleotides atthe 3′ terminus and at least two LNA nucleotides at the 5′ terminus.

Still more preferably the antisense-oligonucleotide of general formula(S4A) has between 12 and 20, more preferably between 13 and 19 and stillmore preferable between 14 and 18 nucleotides and between 2 and 5,preferably 3 and 5 and more preferably between 3 and 4 LNA units at the3′ terminal end and between 2 and 5, preferably 3 and 5 and morepreferably between 3 and 4 LNA units at the 5′ terminal end. Preferablythe antisense-oligonucleotides are GAPmers of the form LNA segment A-DNAsegment-LNA segment B. Preferably the antisense-oligonucleotides containat least 6, more preferably at least 7 and most preferably at least 8non-LNA units such as DNA units in between the two LNA segments.Suitable nucleobases for the non-LNA units and the LNA units aredisclosed in the chapter “Nucleobases”.

Moreover, the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the antisense-oligonucleotide is represented by thefollowing sequence 5′-N⁷-AATGGACC-N⁸-3′ (Seq. ID No. 100), wherein N⁷represents: TGAATCTTGAATATCTCATG-, GAATCTTGAATATCTCATG-,AATCTTGAATATCTCATG-, ATCTTGAATATCTCATG-, TCTTGAATATCTCATG-,CTTGAATATCTCATG-, TTGAATATCTCATG-, TGAATATCTCATG-, GAATATCTCATG-,AATATCTCATG-, ATATCTCATG-, TATCTCATG-, ATCTCATG-, TCTCATG-, CTCATG-,TCATG-, CATG-, ATG-, TG-, or G-;

N⁸

represents: -AGTATTCTAGAAACTCACCA, -AGTATTCTAGAAACTCACC,-AGTATTCTAGAAACTCAC, -AGTATTCTAGAAACTCA, -AGTATTCTAGAAACTC,-AGTATTCTAGAAACT, -AGTATTCTAGAAAC, -AGTATTCTAGAAA, -AGTATTCTAGAA,-AGTATTCTAGA, -AGTATTCTAG, -AGTATTCTA, -AGTATTCT, -AGTATTC, -AGTATT,-AGTAT, -AGTA, -AGT, -AG, or -A;and salts and optical isomers of the antisense-oligonucleotide.

The antisense-oligonucleotides of formula S6 (Seq. ID No. 100)preferably comprise 2 to 10 LNA units, more preferably 3 to 9 LNA unitsand still more preferably 4 to 8 LNA units and also preferably at least6 non-LNA units, more preferably at least 7 non-LNA units and mostpreferably at least 8 non-LNA units. The non-LNA units are preferablyDNA units. The LNA units are preferably positioned at the 3′ terminalend (also named 3′ terminus) and the 5′ terminal end (also named 5′terminus). Preferably at least one and more preferably at least two LNAunits are present at the 3′ terminal end and/or at the 5′ terminal end.

Thus, preferred are antisense-oligonucleotides of the present inventiondesigned as GAPmers which contain 2 to 10 LNA units and which especiallycontain 1 to 5 LNA units at the 5′ terminal end and 1 to 5 LNA units atthe 3′ terminal end of the antisense-oligonucleotide and between the LNAunits at least 7 and more preferably at least 8 DNA units. Morepreferably the antisense-oligonucleotides comprise 2 to 4 LNA units atthe 5′ terminal end and 2 to 4 LNA units at the 3′ terminal end andstill more preferred comprise 3 to 4 LNA units at the 5′ terminal endand 3 to 4 LNA units at the 3′ terminal end and contain preferably atleast 7 non-LNA units and most preferably at least 8 non-LNA units suchas DNA units in between both LNA segments.

Moreover the antisense-oligonucleotides may contain common nucleobasessuch as adenine, guanine, cytosine, thymine and uracil as well as commonderivatives thereof such as 5-methylcytosine or 2-aminoadenine. Theantisense-oligonucleotides of the present invention may also containmodified internucleotide bridges such as phosphorothioate orphosphorodithioate instead of phosphate bridges. Such modifications maybe present only in the LNA segments or only in the non-LNA segment ofthe antisense-oligonucleotide. As LNA units especially the residues b¹to b⁹ as disclosed herein are preferred.

Thus, preferred are antisense-oligonucleotides of the formula (S6):

(Seq. ID No. 100) 5′-N⁷-AATGGACC-N⁸-3′whereinN⁷ represents: TGAATCTTGAATATCTCATG-, GAATCTTGAATATCTCATG-,AATCTTGAATATCTCATG-, ATCTTGAATATCTCATG-, TCTTGAATATCTCATG-,CTTGAATATCTCATG-, TTGAATATCTCATG-, TGAATATCTCATG-, GAATATCTCATG-,AATATCTCATG-, ATATCTCATG-, TATCTCATG-, ATCTCATG-, TCTCATG-, CTCATG-,TCATG-, CATG-, ATG-, TG-, or G-;andN⁸ is selected from: -AGTATTCTAGAAACTCACCA, -AGTATTCTAGAAACTCACC,-AGTATTCTAGAAACTCAC, -AGTATTCTAGAAACTCA, -AGTATTCTAGAAACTC,-AGTATTCTAGAAACT, -AGTATTCTAGAAAC, -AGTATTCTAGAAA, -AGTATTCTAGAA,-AGTATTCTAGA, -AGTATTCTAG, -AGTATTCTA, -AGTATTCT, -AGTATTC, -AGTATT,-AGTAT, -AGTA, -AGT, -AG, or -A.

Preferably the antisense-oligonucleotide of general formula (S6) hasbetween 10 and 28 nucleotides and at least one LNA nucleotide at the 3′terminus and at least one LNA nucleotide at the 5′ terminus. As LNAnucleotides (LNA units) especially these disclosed in the chapter“Locked Nucleic Acids (LNA®)” and preferably in the chapter “PreferredLNAs” are suitable and as internucleotides bridges especially thesedisclosed in the chapter “Internucleotide Linkages (IL)” are suitable.

More preferably the antisense-oligonucleotide of general formula (S6)has between 11 and 24 nucleotides and at least two LNA nucleotides atthe 3′ terminus and at least two LNA nucleotides at the 5′ terminus.

Still more preferably the antisense-oligonucleotide of general formula(S6) has between 12 and 20, more preferably between 13 and 19 and stillmore preferable between 14 and 18 nucleotides and between 2 and 5,preferably 3 and 5 and more preferably between 3 and 4 LNA units at the3′ terminal end and between 2 and 5, preferably 3 and 5 and morepreferably between 3 and 4 LNA units at the 5′ terminal end. Preferablythe antisense-oligonucleotides are GAPmers of the form LNA segment A-DNAsegment-LNA segment B. Preferably the antisense-oligonucleotides containat least 6, more preferably at least 7 and most preferably at least 8non-LNA units such as DNA units in between the two LNA segments.Suitable nucleobases for the non-LNA units and the LNA units aredisclosed in the chapter “Nucleobases”.

Further preferred are antisense-oligonucleotides of the formula (S6):

5′-N⁷-AATGGACC-N⁸-3′whereinN⁷ represents: AATCTTGAATATCTCATG-, ATCTTGAATATCTCATG-,TCTTGAATATCTCATG-, CTTGAATATCTCATG-, TTGAATATCTCATG-, TGAATATCTCATG-,GAATATCTCATG-, AATATCTCATG-, ATATCTCATG-, TATCTCATG-, ATCTCATG-,TCTCATG-, CTCATG-, TCATG-, CATG-, ATG-, TG-, or G-;andN⁸ is selected from: -AGTATTCTAGAAACTCAC, -AGTATTCTAGAAACTCA,-AGTATTCTAGAAACTC, -AGTATTCTAGAAACT, -AGTATTCTAGAAAC, -AGTATTCTAGAAA,-AGTATTCTAGAA, -AGTATTCTAGA, -AGTATTCTAG, -AGTATTCTA, -AGTATTCT,-AGTATTC, -AGTATT, -AGTAT, -AGTA, -AGT, -AG, or -A.

Also preferred are antisense-oligonucleotides of the formula (S6):

5′-N⁷-AATGGACC-N⁸-3′whereinN⁷ represents: TGAATATCTCATG-, GAATATCTCATG-, AATATCTCATG-, ATATCTCATG-,TATCTCATG-, ATCTCATG-, TCTCATG-, CTCATG-, TCATG-, CATG-, ATG-, TG-, orG-;andN⁸ is selected from: -AGTATTCTAGAAA, -AGTATTCTAGAA, -AGTATTCTAGA,-AGTATTCTAG, -AGTATTCTA, -AGTATTCT, -AGTATTC, -AGTATT, -AGTAT, -AGTA,-AGT, -AG, or -A.

Also preferred are antisense-oligonucleotides of the formula (S6):

5′-N⁷-AATGGACC-N⁸-3′whereinN⁷ represents: ATCTCATG-, TCTCATG-, CTCATG-, TCATG-, CATG-, ATG-, TG-,or G-;andN⁸ is selected from: -AGTATTCT, -AGTATTC, -AGTATT, -AGTAT, -AGTA, -AGT,-AG, or -A.

Preferably, the present invention is directed toantisense-oligonucleotide(s) consisting of 12 to 24 nucleotides and atleast three of the 12 to 24 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the antisense-oligonucleotide is represented by thefollowing sequence 5′-N⁷A-GAATGGACCA-N⁸A-3′ (Seq. ID No. 73), wherein

N^(7A) represents: TGAATCTTGAATATCTCAT- (Seq. ID No. 725),GAATCTTGAATATCTCAT- (Seq. ID No. 726), AATCTTGAATATCTCAT- (Seq. ID No.727), ATCTTGAATATCTCAT- (Seq. ID No. 728), TCTTGAATATCTCAT- (Seq. ID No.729), CTTGAATATCTCAT- (Seq. ID No. 730), TTGAATATCTCAT- (Seq. ID No.731), TGAATATCTCAT- (Seq. ID No. 732), GAATATCTCAT- (Seq. ID No. 733),AATATCTCAT- (Seq. ID No. 734), ATATCTCAT-, TATCTCAT-, ATCTCAT-, TCTCAT-,CTCAT-, TCAT-, CAT-, AT-, or T-;N^(8A) represents: -GTATTCTAGAAACTCACCA (Seq. ID No. 735),-GTATTCTAGAAACTCACC (Seq. ID No. 736), -GTATTCTAGAAACTCAC (Seq. ID No.737), -GTATTCTAGAAACTCA (Seq. ID No. 738), -GTATTCTAGAAACTC (Seq. ID No.739), -GTATTCTAGAAACT (Seq. ID No. 740), -GTATTCTAGAAAC (Seq. ID No.741), -GTATTCTAGAAA (Seq. ID No. 742), -GTATTCTAGAA (Seq. ID No. 743),-GTATTCTAGA (Seq. ID No. 744), -GTATTCTAG, -GTATTCTA, -GTATTCT, -GTATTC,-GTATT, -GTAT, -GTA, -G T, or -G;and salts and optical isomers of the antisense-oligonucleotide.

Preferably N^(7A) represents: AATCTTGAATATCTCAT-, ATCTTGAATATCTCAT-,TCTTGAATATCTCAT-, CTTGAATATCTCAT-, TTGAATATCTCAT-, TGAATATCTCAT-,GAATATCTCAT-, AATATCTCAT-, ATATCTCAT-, TATCTCAT-, ATCTCAT-, TCTCAT-,CTCAT-, TCAT-, CAT-, AT-, or T-;

andN⁸A represents: -GTATTCTAGAAACTCAC, -GTATTCTAGAAACTCA, -GTATTCTAGAAACTC,-GTATTCTAGAAACT, -GTATTCTAGAAAC, -GTATTCTAGAAA, -GTATTCTAGAA,-GTATTCTAGA, -GTATTCTAG, -GTATTCTA, -GTATTCT, -GTATTC, -GTATT, -GTAT,-GTA, -GT, or -G.

More preferably N^(7A) represents: TGAATATCTCAT-, GAATATCTCAT-,AATATCTCAT-, ATATCTCAT-, TATCTCAT-, ATCTCAT-, TCTCAT-, CTCAT-, TCAT-,CAT-, AT-, or T-; and

N⁸A represents: -GTATTCTAGAAA, -GTATTCTAGAA, -GTATTCTAGA, -GTATTCTAG,-GTATTCTA, -GTATTCT, -GTATTC, -GTATT, -GTAT, -GTA, -GT, or -G.

Still more preferably N^(7A) represents: ATCTCAT-, TCTCAT-, CTCAT-,TCAT-, CAT-, AT-, or T-; and

N^(8A) represents: -GTATTCT, -GTATTC, -GTATT, -GTAT, -GTA, -GT, or -G.

Preferably the antisense-oligonucleotide of general formula (S6A/Seq. IDNo. 73) has between 12 and 24 nucleotides and at least one LNAnucleotide at the 3′ terminus and at least one LNA nucleotide at the 5′terminus. As LNA nucleotides (LNA units) especially these disclosed inthe chapter “Locked Nucleic Acids (LNA®)” and preferably in the chapter“Preferred LNAs” are suitable and as internucleotides bridges especiallythese disclosed in the chapter “Internucleotide Linkages (IL)” aresuitable.

More preferably the antisense-oligonucleotide of general formula (S6A)has between 12 and 22 nucleotides and at least two LNA nucleotides atthe 3′ terminus and at least two LNA nucleotides at the 5′ terminus.

Still more preferably the antisense-oligonucleotide of general formula(S6A) has between 12 and 20, more preferably between 13 and 19 and stillmore preferable between 14 and 18 nucleotides and between 2 and 5,preferably 3 and 5 and more preferably between 3 and 4 LNA units at the3′ terminal end and between 2 and 5, preferably 3 and 5 and morepreferably between 3 and 4 LNA units at the 5′ terminal end. Preferablythe antisense-oligonucleotides are GAPmers of the form LNA segment A-DNAsegment-LNA segment B. Preferably the antisense-oligonucleotides containat least 6, more preferably at least 7 and most preferably at least 8non-LNA units such as DNA units in between the two LNA segments.Suitable nucleobases for the non-LNA units and the LNA units aredisclosed in the chapter “Nucleobases”.

Moreover, the present invention is directed toantisense-oligonucleotide(s) consisting of 10 to 28 nucleotides and atleast two of the 10 to 28 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the antisense-oligonucleotide is represented by thefollowing sequence 5′-N⁹-ATTAATAA-N¹⁰-3′ (Seq. ID No. 101), wherein

N⁹ represents: ATTCATATTTATATACAGGC-, TTCATATTTATATACAGGC-,TCATATTTATATACAGGC-, CATATTTATATACAGGC-, ATATTTATATACAGGC-,TATTTATATACAGGC-, ATTTATATACAGGC-, TTTATATACAGGC-, TTATATACAGGC-,TATATACAGGC-, ATATACAGGC-, TATACAGGC-, ATACAGGC-, TACAGGC-, ACAGGC-,CAGGC-, AGGC-, GGC-, GC-, or C-;

N¹⁰

represents: -AGTGCAAATGTTATTGGCTA, -AGTGCAAATGTTATTGGCT,-AGTGCAAATGTTATTGGC, -AGTGCAAATGTTATTGG, -AGTGCAAATGTTATTG,-AGTGCAAATGTTATT, -AGTGCAAATGTTAT, -AGTGCAAATGTTA, -AGTGCAAATGTT,-AGTGCAAATGT, -AGTGCAAATG, -AGTGCAAAT, -AGTGCAAA, -AGTGCAA, -AGTGCA,-AGTGC, -AGTG, -AGT, -AG, or -A;and salts and optical isomers of the antisense-oligonucleotide.

The antisense-oligonucleotides of formula S7 (Seq. ID No. 101)preferably comprise 2 to 10 LNA units, more preferably 3 to 9 LNA unitsand still more preferably 4 to 8 LNA units and also preferably at least6 non-LNA units, more preferably at least 7 non-LNA units and mostpreferably at least 8 non-LNA units. The non-LNA units are preferablyDNA units. The LNA units are preferably positioned at the 3′ terminalend (also named 3′ terminus) and the 5′ terminal end (also named 5′terminus). Preferably at least one and more preferably at least two LNAunits are present at the 3′ terminal end and/or at the 5′ terminal end.

Thus, preferred are antisense-oligonucleotides of the present inventiondesigned as GAPmers which contain 2 to 10 LNA units and which especiallycontain 1 to 5 LNA units at the 5′ terminal end and 1 to 5 LNA units atthe 3′ terminal end of the antisense-oligonucleotide and between the LNAunits at least 7 and more preferably at least 8 DNA units. Morepreferably the antisense-oligonucleotides comprise 2 to 4 LNA units atthe 5′ terminal end and 2 to 4 LNA units at the 3′ terminal end andstill more preferred comprise 3 to 4 LNA units at the 5′ terminal endand 3 to 4 LNA units at the 3′ terminal end and contain preferably atleast 7 non-LNA units and most preferably at least 8 non-LNA units suchas DNA units in between both LNA segments.

Moreover the antisense-oligonucleotides may contain common nucleobasessuch as adenine, guanine, cytosine, thymine and uracil as well as commonderivatives thereof such as 5-methylcytosine or 2-aminoadenine. Theantisense-oligonucleotides of the present invention may also containmodified internucleotide bridges such as phosphorothioate orphosphorodithioate instead of phosphate bridges. Such modifications maybe present only in the LNA segments or only in the non-LNA segment ofthe antisense-oligonucleotide. As LNA units especially the residues b¹to b⁹ as disclosed herein are preferred.

Thus, preferred are antisense-oligonucleotides of the formula (S7):

(Seq. ID No. 101) 5′-N⁹-ATTAATAA-N¹⁰-3′whereinN⁹ represents: ATTCATATTTATATACAGGC-, TTCATATTTATATACAGGC-,TCATATTTATATACAGGC-,

CATATTTATATACAGGC-, ATATTTATATACAGGC-, TATTTATATACAGGC-,ATTTATATACAGGC-, TTTATATACAGGC-, TTATATACAGGC-, TATATACAGGC-,ATATACAGGC-, TATACAGGC-, ATACAGGC-, TACAGGC-, ACAGGC-, CAGGC-, AGGC-,GGC-, GC-, or C-;

and N¹⁰ is selected from: -AGTGCAAATGTTATTGGCTA, -AGTGCAAATGTTATTGGCT,-AGTGCAAATGTTATTGGC, -AGTGCAAATGTTATTGG, -AGTGCAAATGTTATTG,-AGTGCAAATGTTATT, -AGTGCAAATGTTAT, -AGTGCAAATGTTA, -AGTGCAAATGTT,-AGTGCAAATGT, -AGTGCAAATG, -AGTGCAAAT, -AGTGCAAA, -AGTGCAA, -AGTGCA,-AGTGC, -AGTG, -AGT, -AG, or -A.

Preferably the antisense-oligonucleotide of general formula (S7) hasbetween 10 and 28 nucleotides and at least one LNA nucleotide at the 3′terminus and at least one LNA nucleotide at the 5′ terminus. As LNAnucleotides (LNA units) especially these disclosed in the chapter“Locked Nucleic Acids (LNA®)” and preferably in the chapter “PreferredLNAs” are suitable and as internucleotides bridges especially thesedisclosed in the chapter “Internucleotide Linkages (IL)” are suitable.

More preferably the antisense-oligonucleotide of general formula (S7)has between 11 and 24 nucleotides and at least two LNA nucleotides atthe 3′ terminus and at least two LNA nucleotides at the 5′ terminus.

Still more preferably the antisense-oligonucleotide of general formula(S7) has between 12 and 20, more preferably between 13 and 19 and stillmore preferable between 14 and 18 nucleotides and between 2 and 5,preferably 3 and 5 and more preferably between 3 and 4 LNA units at the3′ terminal end and between 2 and 5, preferably 3 and 5 and morepreferably between 3 and 4 LNA units at the 5′ terminal end. Preferablythe antisense-oligonucleotides are GAPmers of the form LNA segment A-DNAsegment-LNA segment B. Preferably the antisense-oligonucleotides containat least 6, more preferably at least 7 and most preferably at least 8non-LNA units such as DNA units in between the two LNA segments.Suitable nucleobases for the non-LNA units and the LNA units aredisclosed in the chapter “Nucleobases”.

Further preferred are antisense-oligonucleotides of the formula (S7):

5′-N⁹-ATTAATAA-N¹⁰-3′whereinN⁹ represents: TCATATTTATATACAGGC-, CATATTTATATACAGGC-,ATATTTATATACAGGC-, TATTTATATACAGGC-, ATTTATATACAGGC-, TTTATATACAGGC-,TTATATACAGGC-, TATATACAGGC-, ATATACAGGC-, TATACAGGC-, ATACAGGC-,TACAGGC-, ACAGGC-, CAGGC-, AGGC-, GGC-, GC-, or C-;andN¹⁰ is selected from: -AGTGCAAATGTTATTGGC, -AGTGCAAATGTTATTGG,-AGTGCAAATGTTATTG, -AGTGCAAATGTTATT, -AGTGCAAATGTTAT, -AGTGCAAATGTTA,-AGTGCAAATGTT, -AGTGCAAATGT, -AGTGCAAATG, -AGTGCAAAT, -AGTGCAAA,-AGTGCAA, -AGTGCA, -AGTGC, -AGTG, -AGT, -AG, or -A.

Also preferred are antisense-oligonucleotides of the formula (S7):

5′-N⁹-ATTAATAA-N¹⁰-3′whereinN⁹ represents: TTTATATACAGGC-, TTATATACAGGC-, TATATACAGGC-, ATATACAGGC-,TATACAGGC-, ATACAGGC-, TACAGGC-, ACAGGC-, CAGGC-, AGGC-, GGC-, GC-, orC-; andN¹⁹ is selected from: -AGTGCAAATGTTA, -AGTGCAAATGTT, -AGTGCAAATGT,-AGTGCAAATG, -AGTGCAAAT, -AGTGCAAA, -AGTGCAA, -AGTGCA, -AGTGC, -AGTG,-AGT, -AG, or -A.

Also preferred are antisense-oligonucleotides of the formula (S7):

5′-N⁹-ATTAATAA-N¹⁰-3′whereinN⁹ represents: ATACAGGC-, TACAGGC-, ACAGGC-, CAGGC-, AGGC-, GGC-, GC-,or C-;andN¹⁰ is selected from: -AGTGCAAA, -AGTGCAA, -AGTGCA, -AGTGC, -AGTG, -AGT,-AG, or -A.

Preferably, the present invention is directed toantisense-oligonucleotide(s) consisting of 12 to 24 nucleotides and atleast three of the 12 to 24 nucleotides are LNAs and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the antisense-oligonucleotide is represented by thefollowing sequence 5′-N⁹A-CATTAATAAA-N¹⁰A-3′ (Seq. ID No. 74), whereinN^(9A) represents: ATTCATATTTATATACAGG- (Seq. ID No. 745),TTCATATTTATATACAGG- (Seq. ID No. 746), TCATATTTATATACAGG- (Seq. ID No.747), CATATTTATATACAGG- (Seq. ID No. 748), ATATTTATATACAGG- (Seq. ID No.749), TATTTATATACAGG- (Seq. ID No. 750), ATTTATATACAGG- (Seq. ID No.751), TTTATATACAGG- (Seq. ID No. 752), TTATATACAGG- (Seq. ID No. 753),TATATACAGG- (Seq. ID No. 754), ATATACAGG-, TATACAGG-, ATACAGG-, TACAGG-,ACAGG-, CAGG-, AGG-, GG-, or G-;

N^(10A) represents: -GTGCAAATGTTATTGGCTA (Seq. ID No. 755),-GTGCAAATGTTATTGGCT (Seq. ID No. 756), -GTGCAAATGTTATTGGC (Seq. ID No.757), -GTGCAAATGTTATTGG (Seq. ID No. 758), -GTGCAAATGTTATTG (Seq. ID No.759), -GTGCAAATGTTATT (Seq. ID No. 760), -GTGCAAATGTTAT (Seq. ID No.761), -GTGCAAATGTTA (Seq. ID No. 762), -GTGCAAATGTT (Seq. ID No. 763),-GTGCAAATGT (Seq. ID No. 764), -GTGCAAATG, -GTGCAAAT, -GTGCAAA, -GTGCAA,-GTGCA, -GTGC, -GTG, -GT, or -G;and salts and optical isomers of the antisense-oligonucleotide.

Preferably N^(9A) represents: TCATATTTATATACAGG-, CATATTTATATACAGG-,ATATTTATATACAGG-, TATTTATATACAGG-, ATTTATATACAGG-, TTTATATACAGG-,TTATATACAGG-, TATATACAGG-, ATATACAGG-, TATACAGG-, ATACAGG-, TACAGG-,ACAGG-, CAGG-, AGG-, GG-, or G-; and

N^(10A) represents: -GTGCAAATGTTATTGGC, -GTGCAAATGTTATTGG,-GTGCAAATGTTATTG, -GTGCAAATGTTATT, -GTGCAAATGTTAT, -GTGCAAATGTTA,-GTGCAAATGTT, -GTGCAAATGT, -GTGCAAATG, -GTGCAAAT, -GTGCAAA, -GTGCAA,-GTGCA, -GTGC, -GTG, -GT, or -G.

More preferably N^(9A) represents: TTTATATACAGG-, TTATATACAGG-,TATATACAGG-, ATATACAGG-, TATACAGG-, ATACAGG-, TACAGG-, ACAGG-, CAGG-,AGG-, GG-, or G-; and

N^(10A) represents: -GTGCAAATGTTA, -GTGCAAATGTT, -GTGCAAATGT,-GTGCAAATG, -GTGCAAAT, -GTGCAAA, -GTGCAA, -GTGCA, -GTGC, -GTG, -GT, or-G.

Still more preferably N^(9A) represents: ATACAGG-, TACAGG-, ACAGG-,CAGG-, AGG-, GG-, or G-; and

N^(19A) represents: -GTGCAAA, -GTGCAA, -GTGCA, -GTGC, -GTG, -GT, or -G.

Preferably the antisense-oligonucleotide of general formula (S7A/Seq. IDNo. 74) has between 12 and 24 nucleotides and at least one LNAnucleotide at the 3′ terminus and at least one LNA nucleotide at the 5′terminus. As LNA nucleotides (LNA units) especially these disclosed inthe chapter “Locked Nucleic Acids (LNA®)” and preferably in the chapter“Preferred LNAs” are suitable and as internucleotides bridges especiallythese disclosed in the chapter “Internucleotide Linkages (IL)” aresuitable.

More preferably the antisense-oligonucleotide of general formula (S7A)has between 12 and 22 nucleotides and at least two LNA nucleotides atthe 3′ terminus and at least two LNA nucleotides at the 5′ terminus.

Still more preferably the antisense-oligonucleotide of general formula(S7A) has between 12 and 20, more preferably between 13 and 19 and stillmore preferable between 14 and 18 nucleotides and between 2 and 5,preferably 3 and 5 and more preferably between 3 and 4 LNA units at the3′ terminal end and between 2 and 5, preferably 3 and 5 and morepreferably between 3 and 4 LNA units at the 5′ terminal end. Preferablythe antisense-oligonucleotides are GAPmers of the form LNA segment A-DNAsegment-LNA segment B. Preferably the antisense-oligonucleotides containat least 6, more preferably at least 7 and most preferably at least 8non-LNA units such as DNA units in between the two LNA segments.Suitable nucleobases for the non-LNA units and the LNA units aredisclosed in the chapter “Nucleobases”.

Moreover, the present invention is directed toantisense-oligonucleotide(s) consisting of 8 to 18, preferably 10 to 28nucleotides and at least two of the 8 to 28, preferably 10 to 28nucleotides are LNAs and the antisense-oligonucleotide is capable ofhybridizing with a region of the gene encoding the TGF-R_(II) or with aregion of the mRNA encoding the TGF-R_(II), wherein theantisense-oligonucleotide is represented by the following sequence5′-(N¹³)_(m)-GTAGTGTT-(N¹⁴)_(n)-3′ (Seq. ID No. 99), wherein

N¹³ represents: CCCAGCCTGCCCCAGAAGAGCTATTTG- (Seq. ID No. 793),CCAGCCTGCCCCAGAAGAGCTATTTG- (Seq. ID No. 794),CAGCCTGCCCCAGAAGAGCTATTTG- (Seq. ID No. 795), AGCCTGCCCCAGAAGAGCTATTTG-(Seq. ID No. 796), GCCTGCCCCAGAAGAGCTATTTG- (Seq. ID No. 797),CCTGCCCCAGAAGAGCTATTTG- (Seq. ID No. 798), CTGCCCCAGAAGAGCTATTTG- (Seq.ID No. 799), TGCCCCAGAAGAGCTATTTG- (Seq. ID No. 800),GCCCCAGAAGAGCTATTTG- (Seq. ID No. 801), CCCCAGAAGAGCTATTTG- (Seq. ID No.802), CCCAGAAGAGCTATTTG- (Seq. ID No. 803), CCAGAAGAGCTATTTG- (Seq. IDNo. 804), CAGAAGAGCTATTTG- (Seq. ID No. 805), AGAAGAGCTATTTG- (Seq. IDNo. 806), GAAGAGCTATTTG- (Seq. ID No. 807), AAGAGCTATTTG- (Seq. ID No.808), AGAGCTATTTG- (Seq. ID No. 809), GAGCTATTTG- (Seq. ID No. 810),AGCTATTTG-, GCTATTTG-, CTATTTG-, TATTTG-, ATTTG-, TTTG-, TTG-, TG-, orG-;andN¹⁴ is selected from: -TAGGGAGCCGTCTTCAGGAATCTTCTC (Seq. ID No. 811),-TAGGGAGCCGTCTTCAGGAATCTTCT (Seq. ID No. 812),-TAGGGAGCCGTCTTCAGGAATCTTC (Seq. ID No. 813), -TAGGGAGCCGTCTTCAGGAATCTT(Seq. ID No. 814), -TAGGGAGCCGTCTTCAGGAATCT (Seq. ID No. 815),-TAGGGAGCCGTCTTCAGGAATC (Seq. ID No. 816), -TAGGGAGCCGTCTTCAGGAAT (Seq.ID No. 817), -TAGGGAGCCGTCTTCAGGAA (Seq. ID No. 818),-TAGGGAGCCGTCTTCAGGA (Seq. ID No. 819), -TAGGGAGCCGTCTTCAGG (Seq. ID No.820), -TAGGGAGCCGTCTTCAG (Seq. ID No. 821), -TAGGGAGCCGTCTTCA (Seq. IDNo. 822), -TAGGGAGCCGTCTTC (Seq. ID No. 823), -TAGGGAGCCGTCTT (Seq. IDNo. 824), -TAGGGAGCCGTCT (Seq. ID No. 825), -TAGGGAGCCGTC (Seq. ID No.826), -TAGGGAGCCGT (Seq. ID No. 827), -TAGGGAGCCG (Seq. ID No. 828),-TAGGGAGCC, -TAGGGAGC, -TAGGGAG, -TAGGGA, -TAGGG, -TAGG, -TA G, -TA, or-T;m represents 0 or 1;n represents 0 or 1;and n+m=1 or 2;and salts and optical isomers of the antisense-oligonucleotide.

The antisense-oligonucleotides of formula S5 (Seq. ID No. 99) preferablycomprise 2 to 10 LNA units, more preferably 3 to 9 LNA units and stillmore preferably 4 to 8 LNA units and also preferably at least 6 non-LNAunits, more preferably at least 7 non-LNA units and most preferably atleast 8 non-LNA units. The non-LNA units are preferably DNA units. TheLNA units are preferably positioned at the 3′ terminal end (also named3′ terminus) and the 5′ terminal end (also named 5′ terminus).Preferably at least one and more preferably at least two LNA units arepresent at the 3′ terminal end and/or at the 5′ terminal end.

Thus, preferred are antisense-oligonucleotides of the present inventiondesigned as GAPmers which contain 2 to 10 LNA units and which especiallycontain 1 to 5 LNA units at the 5′ terminal end and 1 to 5 LNA units atthe 3′ terminal end of the antisense-oligonucleotide and between the LNAunits at least 7 and more preferably at least 8 DNA units. Morepreferably the antisense-oligonucleotides comprise 2 to 4 LNA units atthe 5′ terminal end and 2 to 4 LNA units at the 3′ terminal end andstill more preferred comprise 3 to 4 LNA units at the 5′ terminal endand 3 to 4 LNA units at the 3′ terminal end and contain preferably atleast 7 non-LNA units and most preferably at least 8 non-LNA units suchas DNA units in between both LNA segments.

Moreover the antisense-oligonucleotides may contain common nucleobasessuch as adenine, guanine, cytosine, thymine and uracil as well as commonderivatives thereof such as 5-methylcytosine or 2-aminoadenine. Theantisense-oligonucleotides of the present invention may also containmodified internucleotide bridges such as phosphorothioate orphosphorodithioate instead of phosphate bridges. Such modifications maybe present only in the LNA segments or only in the non-LNA segment ofthe antisense-oligonucleotide. As LNA units especially the residues b¹to b⁹ as disclosed herein are preferred.

Thus, preferred are antisense-oligonucleotides of the formula (S5):

5′-(N¹³)_(m)-GTAGTGTT-(N¹⁴)_(n)-3′whereinN¹³ represents: GCCTGCCCCAGAAGAGCTATTTG-CCTGCCCCAGAAGAGCTATTTG-,CTGCCCCAGAAGAGCTATTTG-TGCCCCAGAAGAGCTATTTG-,GCCCCAGAAGAGCTATTTG-CCCCAGAAGAGCTATTTG-, CCCAGAAGAGCTATTTG-,CCAGAAGAGCTATTTG-CAGAAGAGCTATTTG-, AGAAGAGCTATTTG-, GAAGAGCTATTTG-,AAGAGCTATTTG-, AGAGCTATTTG-, GAGCTATTTG-, AGCTATTTG-, GCTATTTG-,CTATTTG-, TATTTG-, ATTTG-, TTTG-, TTG-, TG-, or G-; andN¹⁴ is selected from: -TAGGGAGCCGTCTTC, -TAGGGAGCCGTCTT, -TAGGGAGCCGTCT,-TAGGGAGCCGTC, -TAGGGAGCCGT, -TAGGGAGCCG, -TAGGGAGCC, -TAGGGAGC,-TAGGGAG, -TAGGGA, -TAGGG, -TAGG, -TAG, -TA, or -T; andm represents 0 or 1; n represents 0 or 1; and n+m=1 or 2.

Preferably the antisense-oligonucleotide of general formula (S5) hasbetween 10 and 28 nucleotides and at least one LNA nucleotide at the 3′terminus and at least one LNA nucleotide at the 5′ terminus. As LNAnucleotides (LNA units) especially these disclosed in the chapter“Locked Nucleic Acids (LNA®)” and preferably in the chapter “PreferredLNAs” are suitable and as internucleotides bridges especially thesedisclosed in the chapter “Internucleotide Linkages (IL)” are suitable.

More preferably the antisense-oligonucleotide of general formula (S5)has between 11 and 24 nucleotides and at least two LNA nucleotides atthe 3′ terminus and at least two LNA nucleotides at the 5′ terminus.

Still more preferably the antisense-oligonucleotide of general formula(S5) has between 12 and 20, more preferably between 13 and 19 and stillmore preferable between 14 and 18 nucleotides and between 2 and 5,preferably 3 and 5 and more preferably between 3 and 4 LNA units at the3′ terminal end and between 2 and 5, preferably 3 and 5 and morepreferably between 3 and 4 LNA units at the 5′ terminal end. Preferablythe antisense-oligonucleotides are GAPmers of the form LNA segment A-DNAsegment-LNA segment B. Preferably the antisense-oligonucleotides containat least 6, more preferably at least 7 and most preferably at least 8non-LNA units such as DNA units in between the two LNA segments.Suitable nucleobases for the non-LNA units and the LNA units aredisclosed in the chapter “Nucleobases”.

Further preferred are antisense-oligonucleotides of the formula (S5):

5′-(N¹³)_(m)-GTAGTGTT-(N¹⁴)_(n)-3′whereinN¹³ represents: CCCCAGAAGAGCTATTTG-, CCCAGAAGAGCTATTTG-,CCAGAAGAGCTATTTG-, CAGAAGAGCTATTTG-, AGAAGAGCTATTTG-, GAAGAGCTATTTG-,AAGAGCTATTTG-, AGAGCTATTTG-, GAGCTATTTG-, AGCTATTTG-, GCTATTTG-,CTATTTG-, TATTTG-, ATTTG-, TTTG-, TTG-, TG-, or G-;andN¹⁴ is selected from: -TAGGGAGCCG, -TAGGGAGCC, -TAGGGAGC, -TAGGGAG,-TAGGGA, -TAGGG, -TAGG, -TAG, -TA, or -T; and m represents 0 or 1; nrepresents 0 or 1; and n+m=1 or 2.

Also preferred are antisense-oligonucleotides of the formula (S5):

5′-(N¹³)_(m)-GTAGTGTT-(N¹⁴)_(n)-3′whereinN¹³ represents: GAAGAGCTATTTG-, AAGAGCTATTTG-, AGAGCTATTTG-,GAGCTATTTG-, AGCTATTTG-, GCTATTTG-, CTATTTG-, TATTTG-, ATTTG-, TTTG-,TTG-, TG-, or G-; andN¹⁴ is selected from: -TAGGG, -TAGG, -TAG, -TA, or -T; andm represents 0 or 1; n represents 0 or 1; and n+m=1 or 2.

Also preferred are antisense-oligonucleotides of the formula (S5):

5′-(N¹³)_(m)-GTAGTGTT-(N¹⁴)_(n)-3′whereinN¹³ represents: CAGAAGAGCTATTTG-, AGAAGAGCTATTTG-, GAAGAGCTATTTG-,AAGAGCTATTTG-, AGAGCTATTTG-, GAGCTATTTG-, AGCTATTTG-, GCTATTTG-,CTATTTG-, TATTTG-, ATTTG-, TTTG-, TTG-, TG-, or G-; andN¹⁴ is selected from: -TAGGGAGCCGTCTTCAGGAATCT, -TAGGGAGCCGTCTTCAGGAATC,-TAGGGAGCCGTCTTCAGGAAT, -TAGGGAGCCGTCTTCAGGAA, -TAGGGAGCCGTCTTCAGGA,-TAGGGAGCCGTCTTCAGG, -TAGGGAGCCGTCTTCAG, -TAGGGAGCCGTCTTCA,-TAGGGAGCCGTCTTC, -TAGGGAGCCGTCTT, -TAGGGAGCCGTCT, -TAGGGAGCCGTC,-TAGGGAGCCGT, -TAGGGAGCCG, -TAGGGAGCC, -TAGGGAGC, -TAGGGAG, -TAGGGA,-TAGGG, -TAGG, -TAG, -TA, or -T; andm represents 0 or 1; n represents 0 or 1; and n+m=1 or 2.

Also preferred are antisense-oligonucleotides of the formula (S5):

5′-(N¹³)_(m)-GTAGTGTT-(N¹⁴)_(n)-3′whereinN¹³ represents: GAGCTATTTG-, AGCTATTTG-, GCTATTTG-, CTATTTG-, TATTTG-,ATTTG-, TTTG-, TTG-, TG-, or G-; and

N¹⁴ is selected from: -TAGGGAGCCGTCTTCAGG, -TAGGGAGCCGTCTTCAG,-TAGGGAGCCGTCTTCA, -TAGGGAGCCGTCTTC, -TAGGGAGCCGTCTT, -TAGGGAGCCGTCT,-TAGGGAGCCGTC, -TAGGGAGCCGT, -TAGGGAGCCG, -TAGGGAGCC, -TAGGGAGC,-TAGGGAG, -TAGGGA, -TAGGG, -TAGG, -TAG, -TA, or -T; and

m represents 0 or 1; n represents 0 or 1; and n+m=1 or 2.

Also preferred are antisense-oligonucleotides of the formula (S5):

5′-(N¹³)_(m)-GTAGTGTT-(N¹⁴)_(n)-3′whereinN¹³ represents: ATTTG-, TTTG-, TTG-, TG-, or G-; andN¹⁴ is selected from: -TAGGGAGCCGTCT, -TAGGGAGCCGTC, -TAGGGAGCCGT,-TAGGGAGC CG, -TAGGGAGCC, -TAGGGAGC, -TAGGGAG, -TAGGGA, -TAGGG, -TAGG,-TAG, -TA, or -T; andm represents 0 or 1; n represents 0 or 1; and n+m=1 or 2.

Preferably the antisense-oligonucleotide of general formula (S5/Seq. IDNo. 99) has between 12 and 24 nucleotides and at least one LNAnucleotide at the 3′ terminus and at least one LNA nucleotide at the 5′terminus. As LNA nucleotides (LNA units) especially these disclosed inthe chapter “Locked Nucleic Acids (LNA®)” and preferably in the chapter“Preferred LNAs” are suitable and as internucleotides bridges especiallythese disclosed in the chapter “Internucleotide Linkages (IL)” aresuitable.

More preferably the antisense-oligonucleotide of general formula (S5)has between 12 and 22 nucleotides and at least two LNA nucleotides atthe 3′ terminus and at least two LNA nucleotides at the 5′ terminus.

Still more preferably the antisense-oligonucleotide of general formula(S5) has between 12 and 20, more preferably between 13 and 19 and stillmore preferable between 14 and 18 nucleotides and between 2 and 5,preferably 3 and 5 and more preferably between 3 and 4 LNA units at the3′ terminal end and between 2 and 5, preferably 3 and 5 and morepreferably between 3 and 4 LNA units at the 5′ terminal end. Preferablythe antisense-oligonucleotides are GAPmers of the form LNA segment A-DNAsegment-LNA segment B. Preferably the antisense-oligonucleotides containat least 6, more preferably at least 7 and most preferably at least 8non-LNA units such as DNA units in between the two LNA segments.Suitable nucleobases for the non-LNA units and the LNA units aredisclosed in the chapter “Nucleobases”.

Another aspect of the present invention relates toantisense-oligonucleotide(s) having a length of 10 to 28 nucleotides,preferably 10 to 24 nucleotides, more preferably 11 to 22 nucleotides or12 to 20 nucleotides, still more preferably 13 to 19 nucleotides, andmost preferably 14 to 18 nucleotides, wherein at least two of thenucleotides, preferably at least three of the nucleotides, and morepreferably at least four of the nucleotides are LNAs and the sequence ofthe antisense-oligonucleotide of the 10 to 28 nucleotides, preferably 10to 24 nucleotides, more preferably 11 to 22 nucleotides or 12 to 20nucleotides, still more preferably 13 to 19 nucleotides, and mostpreferably 14 to 18 nucleotides is selected from the group of sequencesof 10 to 28 nucleotides, preferably 10 to 24 nucleotides, morepreferably 11 to 22 nucleotides or 12 to 20 nucleotides, still morepreferably 13 to 19 nucleotides, and most preferably 14 to 18nucleotides contained in a sequence selected from the following group:

(Seq. ID No. 75: 383-423 of Seq. ID No. 1)GAATCTTGAATATCTCATGAATGGACCAGTATTCTAGAAAC, (Seq. ID No. 77: 2245-2285 ofSeq. ID No. 1) TTCATATTTATATACAGGCATTAATAAAGTGCAAATGTTAT, (Seq. ID No.78: 2315-2356 of Seq. ID No. 1)TGAGGAAGTGCTAACACAGCTTATCCTATGACAATGTCAAAG, (Seq. ID No. 79: 2528-2576of Seq. ID No. 1) GCCTGCCCCAGAAGAGCTATTTGGTAGTGTTTAGGGAGCCGTCTTCAGG,(Seq. ID No. 81: 3205-3253 of Seq. ID No. 1)CGCAGGTCCTCCCAGCTGATGACATGCCGCGTCAGGTACTCCTGTAGGT, (Seq. ID No. 83:4141-4218 of Seq. ID No. 1)ATGTCGTTATTAACCGACTTCTGAACGTGCGGTGGGATCGTGCTGGCGATACGCGTCCACAGGACGATGTGCAGCGGC, (Seq. ID No. 84: 4216-4289 of Seq. IDNo. 1) GGCCACAGGCCCCTGAGCAGCCCCCGACCCATGGCAGACCCCGCTGCTCGTCATAGACCGAGCCCCCAGCGCAG, (Seq. ID No. 86: 4141-4289 of Seq. IDNo. 1) ATGTCGTTATTAACCGACTTCTGAACGTGCGGTGGGATCGTGCTGGCGATACGCGTCCACAGGACGATGTGCAGCGGCCACAGGCCCCTGAGCAGCCCCCGACCCATGGCAGACCCCGCTGCTCGTCATAGACCGAGCCCCCA GCGCAG, (Seq. ID No. 87:388-418 of Seq. ID No. 1) TTGAATATCTCATGAATGGACCAGTATTCTA, (Seq. ID No.88: 483-515 of Seq. ID No. 1) CAAGTGGAATTTCTAGGCGCCTCTATGCTACTG, (Seq.ID No. 89: 2250-2280 of Seq. ID No. 1) ATTTATATACAGGCATTAATAAAGTGCAAAT,(Seq. ID No. 90: 2320-2351 of Seq. ID No. 1)AAGTGCTAACACAGCTTATCCTATGACAATGT, (Seq. ID No. 91: 2533-2571 of Seq. IDNo. 1) CCCCAGAAGAGCTATTTGGTAGTGTTTAGGGAGCCGTCT, (Seq. ID No. 92:2753-2830 of Seq. ID No. 1)CTGGTCGCCCTCGATCTCTCAACACGTTGTCCTTCATGCTTTCGACACAGGGGTGCTCCCGCACCTTGGAACCAAATG, (Seq. ID No. 93: 3210-3248 of Seq. IDNo. 1) GTCCTCCCAGCTGATGACATGCCGCGTCAGGTACTCCTG, (Seq. ID No. 94:3655-3694 of Seq. ID No. 1) CTCAGCTTCTGCTGCCGGTTAACGCGGTAGCAGTAGAAGA,(Seq. ID No. 95: 4146-4213 of Seq. ID No. 1)GTTATTAACCGACTTCTGAACGTGCGGTGGGATCGTGCTGGCGATA CGCGTCCACAGGACGATGTGCA,(Seq. ID No. 96: 4221-4284 of Seq. ID No. 1)CAGGCCCCTGAGCAGCCCCCGACCCATGGCAGACCCCGCTGCTCGTCA TAGACCGAGCCCCCAG, (Seq.ID No. 97: 4495-4546 of Seq. ID No. 1)CACGCGCGGGGGTGTCGTCGCTCCGTGCGCGCGAGTGACTCACTCAAC TTCA,wherein the antisense-oligonucleotide is capable of selectivelyhybridizing in regard to the whole human transcriptome only with thegene encoding TGF-R_(II) or with the mRNA encoding TGF-R_(II) and saltsand optical isomers of said antisense-oligonucleotide.

Said antisense-oligonucleotide having a sequence contained in thesequences No. 75, 77, 78, 79, 81, 83, 84, 86 97 have between 2 and 5,preferably 3 and 5 and more preferably between 3 and 4 LNA units at the3′ terminal end and between 2 and 5, preferably 3 and 5 and morepreferably between 3 and 4 LNA units at the 5′ terminal end and havepreferably the structure of GAPmers of the form LNA segment A-DNAsegment-LNA segment B. As LNA nucleotides (LNA units) especially thesedisclosed in the chapter “Locked Nucleic Acids (LNA®)” and preferably inthe chapter “Preferred LNAs” are suitable and as internucleotidesbridges especially these disclosed in the chapter “InternucleotideLinkages (IL)” are suitable. Preferably said antisense-oligonucleotidescontain at least 6, more preferably at least 7 and most preferably atleast 8 non-LNA units such as DNA units in between the two LNA segments.Suitable nucleobases for the non-LNA units and the LNA units aredisclosed in the chapter “Nucleobases”. Suitable examples for saidantisense-oligonucleotides are represented by the formulae (51) to (S7),(S1A) to (S4A), (S6A) and (S7A).

The Seq. ID No. 1 represents the antisense strand of the cDNA (cDNA)(5′-3′ antisense-sequence) of the Homo sapiens transforming growthfactor, beta receptor II (TGF-R_(II)), transcript variant 2.

The Seq. ID No. 2 represents the sense strand of the cDNA (5′-3′sense-sequence) of the Homo sapiens transforming growth factor, betareceptor II (70/80 kDa) (TGF-R_(II)), transcript variant 2.Alternatively, one can also regard the sequence of Seq. ID No. 2 torepresent the sequence of the mRNA of the Homo sapiens transforminggrowth factor, beta receptor II (TGF-R_(II)), transcript variant 2 (Seq.ID No. 3), but written in the DNA code, i.e. represented in G, C, A, Tcode, and not in the RNA code.

The Seq. ID No. 3 represents the mRNA (5′-3′ sense-sequence) of the Homosapiens transforming growth factor, beta receptor II (TGF-R_(II)),transcript variant 2. It is evident that the mRNA displayed in Seq. IDNo. 3 is written in the RNA code, i.e.

represented in G, C, A, U code.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

It shall be understood, that “coding DNA strand”, as used herein, refersto the DNA strand that is identical to the mRNA (except that is writtenin the DNA code) and that encompasses the codons that used for proteintranslation. It is not used as template for the transcription into mRNA.Thus, the terms “coding DNA strand”, “sense DNA strand” and“non-template DNA strand” can be used interchangeably. Furthermore,“non-coding DNA strand”, as used herein, refers to the DNA strand thatis complementary to the “coding DNA strand” and serves as a template forthe transcription of mRNA. Thus, the terms “non-coding DNA strand”,“antisense DNA strand” and “template DNA strand” can be usedinterchangeably

The term “antisense-oligonucleotide” refers to an oligomer or polymer ofribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics orvariants thereof such as antisense-oligonucleotides having a modifiedinternucleotide linkage like a phosphorothioate linkage and/or one ormore modified nucleobases such as 5-methylcytosine and/or one or moremodified nucleotide units such as LNAs like β-D-oxy-LNA. The term“antisense-oligonucleotide” includes oligonucleotides composed ofnaturally-occurring nucleobases, sugars and covalent internucleotide(backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native forms,because of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases. The antisense-oligonucleotidesare short catalytic RNAs or catalytic oligonucleotides which hybridizeto the target nucleic acid and inhibit its expression.

The term “nucleoside” is well known to a skilled person and refers to apentose sugar moiety like ribose, desoxyribose or a modified or lockedribose or a modified or locked desoxyribose like the LNAs which arebelow disclosed in detail. A nucleobase is linked to the glycosidiccarbon atom (position 1′ of the pentose) and an internucleotide linkageis formed between the 3′ oxygen or sulfur atom and preferably the 3′oxygen atom of a nucleoside and the 5′ oxygen or sulfur atom andpreferably the 5′ oxygen atom of the adjacent nucleoside, while theinternucleotide linkage does not belong to the nucleoside (see FIG. 2).

The term “nucleotide” is well known to a skilled person and refers to apentose sugar moiety like ribose, desoxyribose or a modified or lockedribose or a modified or locked desoxyribose like the LNAs which arebelow disclosed in detail. A nucleobase is linked to the glycosidiccarbon atom (position 1′ of the pentose) and an internucleotide linkageis formed between the 3′ oxygen or sulfur atom and preferably the 3′oxygen atom of a nucleotide and the 5′ oxygen or sulfur atom andpreferably the 5′ oxygen atom of the adjacent nucleotide, while theinternucleotide linkage is a part of the nucleotide (see FIG. 2).

Nucleobases

The term “nucleobase” is herein abbreviated with “B” and refers to thefive standard nucleotide bases adenine (A), thymine (T), guanine (G),cytosine (C), and uracil (U) as well as to modifications or analoguesthereof or analogues with ability to form Watson-Crick base pair withbases in the complimentary strand. Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (C*),5-hydroxymethyl cytosine, N⁴-methylcytosine, xanthine, hypoxanthine,7-deazaxanthine, 2-aminoadenine, 6-methyladenine, 6-methylguanine,6-ethyladenine, 6-ethylguanine, 2-propyladenine, 2-propylguanine,6-carboxyuracil, 5-halouracil, 5,6-dihydrouracil, 5-halocytosine,5-propynyl uracil, 5-propynyl cytosine, 6-aza uracil, 6-aza cytosine,6-aza thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-fluoroadenine,8-chloroadenine, 8-bromoadenine, 8-iodoadenine, 8-aminoadenine,8-thioladenine, 8-thioalkyladenine, 8-hydroxyladenine, 8-fluoroguanine,8-chloroguanine, 8-bromoguanine, 8-iodoguanine, 8-aminoguanine,8-thiolguanine, 8-thioalkylguanine, 8-hydroxylguanine, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, 5-trifluoromethyluracil,5-fluorocytosine, 5-bromocytosine, 5-chlorocytosine, 5-iodocytosine,5-trifluoromethylcytosine, 7-methylguanine, 7-methyladenine,8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine,7-deaza-8-azaadenine, 3-deazaguanine, 3-deazaadenine, 2-thiouracil,2-thiothymine and 2-thiocytosine etc., with 5-methylcytosine and/or2-aminoadenine substitutions being preferred since these modificationshave been shown to increase nucleic acid duplex stability.

Preferred antisense-oligonucleotides of the present invention cancomprise analogues of nucleobases. The nucleobase of only one nucleotideunit of the antisense-oligonucleotide could be replaced by an analogueof a nucleobase or two, three, four, five or even all nucleobases in anantisense-oligonucleotide could be replaced by analogues of nucleobases,such as 5-methylcytosine, or N⁶-methyladenine or 2-aminoadenine.Preferably the LNA units might be connected to analogues of nucleobasessuch as 5-methylcytosine.

It will be recognized that when referring to a sequence of nucleotidesor monomers, what is referred to, is the sequence of bases, such as A,T, G, C or U. However, except the specific examples disclosed in Tables3 to 8 the representation of the antisense-oligonucleotides by theletter code A, T, G, C and U has to be understood that saidantisense-oligonucleotide may contain any the nucleobases as disclosedherein, any of the 3′ end groups as disclosed herein, any of the 5′ endgroups as disclosed herein, and any of the internucleotide linkages(also referred to as internucleotide bridges) as disclosed herein. Thenucleotides A, T, G, C and U have also to be understood as being LNAnucleotides or non-LNA nucleotides such as preferably DNA nucleotides.

Only in regard to the specific examples as disclosed in Tables 4 to 9the nucleobases, the LNA units, the non-LNA units, the internucleotidelinkages and the end groups are further specified as outlined in thechapter “Legend” before Table 2.

The antisense-oligonucleotides as well as the salts of theantisense-oligonucleotides as disclosed herein have been proven to becomplementary to the target which is the gene encoding for theTGF-R_(II) or the mRNA encoding the TGF-R_(II), i.e., hybridizesufficiently well and with sufficient specificity and especiallyselectivity to give the desired inhibitory effect.

The term “salt” refers to physiologically and/or pharmaceuticallyacceptable salts of the antisense-oligonucleotides of the presentinvention. The antisense-oligonucleotides contain nucleobases likeadenine, guanine, thymine, cytosine or derivatives thereof which arebasic and which form a salt like a chloride or mesylate salt. Theinternucleotide linkage preferably contains a negatively charged oxygenor sulfur atom which form salts like the sodium, lithium or potassiumsalt. Thus, pharmaceutically acceptable base addition salts are formedwith inorganic bases or organic bases. Examples for suitable organic andinorganic bases are bases derived from metal ions, e.g., aluminum,alkali metal ions, such as sodium or potassium, alkaline earth metalions such as calcium or magnesium, or an amine salt ion or alkali- oralkaline-earth hydroxides, -carbonates or -bicarbonates. Examplesinclude aqueous LiOH, NaOH, KOH, NH₄OH, potassium carbonate, ammonia andsodium bicarbonate, ammonium salts, primary, secondary and tertiaryamines, such as, e.g., tetraalkylammonium hydroxide, lower alkylaminessuch as methylamine, t-butylamine, procaine, ethanolamine,arylalkylamines such as dibenzylamine and N,N-dibenzylethylenediamine,lower alkylpiperidines such as N-ethylpiperidine, cycloalkylamines suchas cyclohexylamine or dicyclohexylamine, morpholine, glucamine,N-methyl- and N,N-dimethylglucamine, 1-adamantylamine, benzathine, orsalts derived from amino acids like arginine, lysine, ornithine oramides of originally neutral or acidic amino acids, chloroprocaine,choline, procaine or the like.

Since the antisense-oligonucleotides are basic, they formpharmaceutically acceptable salts with organic and inorganic acids.Examples of suitable acids for such acid addition salt formation arehydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid,acetic acid, citric acid, oxalic acid, malonic acid, salicylic acid,p-aminosalicylic acid, malic acid, fumaric acid, succinic acid, ascorbicacid, maleic acid, sulfonic acid, phosphonic acid, perchloric acid,nitric acid, formic acid, propionic acid, gluconic acid, lactic acid,tartaric acid, hydroxymaleic acid, pyruvic acid, phenylacetic acid,benzoic acid, p-aminobenzoic acid, p-hydroxybenzoic acid,methanesulfonic acid, ethanesulfonic acid, nitrous acid,hydroxyethanesulfonic acid, ethylenesulfonic acid, p-toluenesulfonicacid, naphthylsulfonic acid, sulfanilic acid, camphersulfonic acid,china acid, mandelic acid, o-methylmandelic acid,hydrogen-benzenesulfonic acid, picric acid, adipic acid,D-o-tolyltartaric acid, tartronic acid, -toluic acid, (o, m, p)-toluicacid, naphthylamine sulfonic acid, and other mineral or carboxylic acidswell known to those skilled in the art. The salts are prepared bycontacting the free base form with a sufficient amount of the desiredacid to produce a salt in the conventional manner.

In the context of this invention, “hybridization” means nucleic acidhybridization, wherein a single-stranded nucleic acid (DNA or RNA)interacts with another single-stranded nucleic acid having a verysimilar or even complementary sequence. Thereby the interaction takesplace by hydrogen bonds between specific nucleobases (base pairing).

As used herein, the term “complementarity” (DNA and RNA base paircomplementarity) refers to the capacity for precise pairing between twonucleic acids. The nucleotides in a base pair are complementary whentheir shape allows them to bond together by hydrogen bonds. Therebyforms the pair of adenine and thymidine (or uracil) two hydrogen bondsand the cytosine-guanine pair forms three hydrogen bonds. “Complementarysequences” as used herein means DNA or RNA sequences, being such thatwhen they are aligned antiparallel to each other, the nucleotide basesat each position in the sequences will be complementary, much likelooking in the mirror and seeing the reverse of things.

The term “specifically hybridizable” as used herein indicates asufficient degree of complementarity or precise base pairing of theantisense-oligonucleotide to the target sequence such that stable andspecific binding occurs between the antisense-oligonucleotide and theDNA or RNA target. The sequence of an oligonucleotide according to theinvention does not need to be 100% complementary to that of its targetnucleic acid to be specifically hybridizable, although a 100%complementarity is preferred. Thereby “100% complementarity” means thatthe antisense-oligonucleotide hybridizes with the target over itscomplete or full length without mismatch. In other words, within thepresent invention it is defined that an antisense compound isspecifically hybridizable when binding of the compound to the target DNAor RNA molecule takes place under physiological or pathologicalconditions but non-specific binding of the antisense-oligonucleotide tonon-target sequences is highly unlikely or even impossible.

Therefore, the present invention refers preferably to antisenseoligonucleotides, wherein the antisense oligonucleotides bind with 100%complementarity to the mRNA encoding TGF RII and do not bind to anyother region in the complete human transcriptome. Further preferred thepresent invention refers to antisense oligonucleotides, wherein theantisense oligonucleotides have 100% complementarity over their completelength to the mRNA encoding TGF RII and have no off-target effects.Alternatively, the present invention refers preferably to antisenseoligonucleotides having 100% complementarity to the mRNA encoding TGFRII but no complementarity to another mRNA of the human transcriptome.Thereby the term “human transcriptome” refers to the total set oftranscripts in the human organism, which means transcripts of all celltypes and environmental conditions (at any given time).

Specificity

The antisense-oligonucleotides of the present invention have in commonthat they are specific in regard to the region where they bind to thegene or to the mRNA encoding TGF-R_(II). According to the presentinvention it is preferred that within the human transcriptome, theantisense-oligonucleotides have 100% complementarity over their fulllength only with the mRNA encoding TGF-RII. In addition, it was a goalof the present invention to find antisense-oligonucleotides withoutcross-reactivity within to the transcriptome of mammalian other thanmonkeys; in particular, the antisense-oligonucleotides have onlycross-reactivity with the transcriptome of great apes. This should avoidoff-effects. Thus the antisense-oligonucleotides of the presentinvention are highly specific concerning hybridization with the gene orwith the mRNA encoding TGF-RII. The antisense-oligonucleotides of theinvention bind preferably over their complete length with 100%complementarity specific to the gene encoding TGF-RII or to the mRNAencoding TGF-RII and do not bind to any other region in the completehuman transcriptome. This means, the antisense-oligonucleotides of thepresent invention hybridize with the target (TGF-RII mRNA) withoutmismatch.

The term “mRNA”, as used herein, may encompass both mRNA containingintrons (also referred to as Pre-mRNA) as well as mRNA which does notcontain any introns.

The antisense-oligonucleotides of the present invention are able to bindor hybridize with the Pre-mRNA and/or with the mRNA. That means theantisense-oligonucleotides can bind to or hybridize at an intron regionor within an intron region of the Pre-mRNA or can bind to or hybridizeat an overlapping intron exon region of the Pre-mRNA or can bind to orhybridize at an exon region or within an exon region of the Pre-mRNA andthe exon region of the mRNA (see FIG. 1). Preferred areantisense-oligonucleotides which are able to bind to or hybridize withPre-mRNA and mRNA. Binding or hybridization of theantisense-oligonucleotides (ASO) to the Pre-mRNA inhibits the 5′ capformation, inhibits splicing of the Pre-mRNA in order to obtain the mRNAand activates RNase H which cleaves the Pre-mRNA. Binding orhybridization of the antisense-oligonucleotides (ASO) to the mRNAactivates RNase H which cleaves the mRNA and inhibits binding of theribosomal subunits.

The antisense-oligonucleotides of the present invention consist of atleast 10 and no more than 28, preferably no more than 24 and morepreferably no more than 20 nucleotides and consequently consist of 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, preferably of 11to 20, or 11 to 19, or 12 to 19, or 13 to 19, or 13 to 18 nucleotidesand more preferably of 14 to 18 nucleotides, wherein at least two,preferably three of these nucleotides are locked nucleic acids (LNA).Shorter antisense-oligonucleotides, i.e. antisense-oligonucleotideshaving less than 10 nucleotides, are also possible but the shorter theantisense-oligonucleotides the higher the risk that the hybridization isnot sufficiently strong anymore and that selectivity will decrease orwill get lost. Non-selective antisense-oligonucleotides bear the risk tobind to undesired regions in the human transcriptome and to undesiredmRNAs coding for other proteins than TGF-R_(II) thereby causingundesired side effects. Longer antisense-oligonucleotides having morethan 20 nucleotides are also possible but further increasing the lengthmake the synthesis of such antisense-oligonucleotides even morecomplicated and expensive without any further benefit in increasingselectivity or strength of hybridization or better stability in regardto degradation.

Thus the present invention is directed to antisense-oligonucleotidesconsisting of 10 to 20 nucleotides, wherein at least two nucleotides andpreferably the 3′ and 5′ terminal nucleotides are LNAs. Thus, it ispreferred that at least the terminal 3′ nucleotide is an LNA and also atleast the 5′ terminal nucleotide is an LNA. In case more than 2 LNAs arepresent, it is preferred that the further LNAs are linked to the 3′ or5′ terminal LNA like it is the case in gapmers as disclosed herein.

One nucleotide building block present in an antisense-oligonucleotide ofthe present invention can be represented by the following generalformula (B1) and (B2):

whereinB represents a nucleobase;IL′ represents —X″—P(═X′)(X⁻)—;R represents —H, —F, —OH, —NH₂, —OCH₃, —OCH₂CH₂OCH₃ and R^(#) represents—H; or R and R^(#) form together the bridge —R^(#)—R— which is selectedfrom —CH₂—O—, —CH₂—S—, —CH₂—NH—, —CH₂—N(CH₃)—, —CH₂—N(C₂H₅)—,—CH₂—CH₂—O—, —CH₂—CH₂—S—, —CH₂—CH₂—NH—, —CH₂—CH₂—N(CH₃)—, or—CH₂—CH₂—N(C₂H₅)—;X′ represents ═O or ═S;X⁻ represents —O⁻, —OH, —OR^(H), —NHR^(H), —N(R^(H))₂, —OCH₂CH₂OR^(H),—OCH₂CH₂SR^(H), —BH₃ ⁻, —R^(H), —SH, —SR^(H), or —S⁻;X″ represents —O—, —NH—, —NR^(H)—, —CH₂—, or —S—;Y is —O—, —NH—, —NR^(H)—, —CH₂— or —S—;R^(H) is selected from hydrogen and C₁₋₄-alkyl and preferably —CH₃ or—C₂H₅ and most preferably —CH₃.

Preferably X⁻ represents —O⁻, —OH, —OCH₃, —NH(CH₃), —N(CH₃)₂,—OCH₂CH₂OCH₃, —OCH₂CH₂SCH₃, —BH₃ ⁻, —CH₃, —SH, —SCH₃, or —S⁻; and morepreferably —O⁻, —OH, —OCH₃, —N(CH₃)₂, —OCH₂CH₂OCH₃, —BH₃ ⁻, —SH, —SCH₃,or —S⁻.

IL′ represents preferably —O—P(O)(O⁻)—, —O—P(O)(S⁻)—, —O—P(S)(S⁻)—,—S—P(O)(O⁻)—, —S—P(O)(S⁻)—, —S—P(S)(S⁻)—, —O—P(O)(O⁻)—, —O—P(O)(S⁻)—,—S—P(O)(O⁻)—, —O—P(O)(R^(H))—, —O—P(O)(OR^(H))—, —O—P(O)(NHR^(H))—,—O—P(O)[N(R^(H))₂]—, —O—P(O)(BH₃ ⁻)—, —O—P(O)(OCH₂CH₂OR^(H))—,—O—P(O)(OCH₂CH₂SR^(H))—, —O—P(O)(O⁻)—, —NR^(H)—P(O)(O⁻)—, wherein R^(H)is selected from hydrogen and C₁₋₄-alkyl.

The group —O—P(O)(R^(H))—O— is preferably —O—P(O)(CH₃)—O— or—O—P(O)(C₂H₅)—O— and most preferably —O—P(O)(CH₃)—O—.

The group —O—P(O)(OR^(H))—O— is preferably —O—P(O)(OCH₃)—O— or—O—P(O)(OC₂H₅)—O— and most preferably —O—P(O)(OCH₃)—O—.

The group —O—P(O)(NHR^(H))—O— is preferably —O—P(O)(NHCH₃)—O— or—O—P(O)(NHC₂H₅)—O— and most preferably —O—P(O)(NHCH₃)—O—.

The group —O—P(O)[N(R^(H))₂]—O— is preferably —O—P(O)[N(CH₃)₂]—O— or—O—P(O)[N(C₂H₅)₂]—O— and most preferably —O—P(O)[N(CH₃)₂]—O—.

The group —O—P(O)(OCH₂CH₂OR^(H))—O— is preferably—O—P(O)(OCH₂CH₂OCH₃)—O— or —O—P(O)(OCH₂CH₂OC₂H₅)—O— and most preferably—O—P(O)(OCH₂CH₂OCH₃)—O—.

The group —O—P(O)(OCH₂CH₂SR^(H))—O— is preferably—O—P(O)(OCH₂CH₂SCH₃)—O— or —O—P(O)(OCH₂CH₂SC₂H₅)—O— and most preferably—O—P(O) (OCH₂CH₂SCH₃)—O—.

The group —O—P(O)(O⁻)—NR^(H)— is preferably —O—P(O)(O⁻)—NH— or—O—P(O)(O⁻)—N(CH₃)— and most preferably —O—P(O)(O⁻)—NH—.

The group —NR^(H)—P(O)(O⁻)—O— is preferably —NH—P(O)(O⁻)—O— or—N(CH₃)—P(O)(O⁻)—O— and most preferably —NH—P(O)(O⁻)—O—.

Even more preferably IL′ represents —O—P(O)(O⁻)—, —O—P(O)(S⁻)—,—O—P(S)(S⁻)—, —O—P(O)(NHR^(H))—, or —O—P(O)[N(R^(H))₂]—, and still morepreferably IL′ represents —O—P(O)(O⁻)—, —O—P(O)(S⁻)—, or —O—P(S)(S⁻)—,and most preferably IL′ represents —O—P(O)(S⁻)—, or —O—P(S)(S⁻)—.

Preferably Y represents —O—.

Preferably B represents a standard nucleobase selected from A, T, G, C,U.

Preferably IL represents —O—P(═O)(S⁻)— or —O—P(═S)(S⁻)—.

The above definitions of B, Y and IL′ apply also to the formula b¹ tob⁹.

Thus the following general formula (B3) to (B6) are preferred:

whereinB represents a nucleobase and preferably A, T, G, C, U;R represents —H, —F, —OH, —NH₂, —N(CH₃)₂, —OCH₃, —OCH₂CH₂OCH₃,—OCH₂CH₂CH₂OH, —OCH₂CH₂CH₂NH₂ and preferably —H;R* represents the moiety —R^(#)—R— as defined below and is, forinstance, preferably selected from —C(R^(a)R^(b))—O—,—C(R^(a)R^(b))—NR^(c)—, —C(R^(a)R^(b))—S—, and—C(R^(a)R^(b))—C(R^(a)R^(b))—O—, wherein the substituents R^(a), R^(b)and R^(c) have the meanings as defined herein. More preferably R* isselected from —CH₂—O—, —CH₂—S—, —CH₂—NH—, —CH₂—N(CH₃)—, —CH₂—CH₂—O—, or—CH₂—CH₂—S—, and more preferably —CH₂—O—, —CH₂—S—, —CH₂—CH₂—O—, or—CH₂—CH₂—S—, and still more preferably —CH₂—O—, —CH₂—S—, or —CH₂—CH₂—O—,and still more preferably —CH₂—O— or —CH₂—S—, and most preferably—CH₂—O—.

Examples of preferred nucleotides which are non-LNA units are thefollowing:

Internucleotide Linkages (IL)

The monomers of the antisense-oligonucleotides described herein arecoupled together via an internucleotide linkage. Suitably, each monomeris linked to the 3′ adjacent monomer via an internucleotide linkage. Theperson having ordinary skill in the art would understand that, in thecontext of the present invention, the 5′ monomer at the end of anoligomer does not comprise a 5′ internucleotide linkage, although it mayor may not comprise a 5′ terminal group. The term “internucleotidelinkage” is intended to mean a group capable of covalently couplingtogether two nucleotides, two nucleotide analogues like two LNAs, and anucleotide and a nucleotide analogue like an LNA. Specific and preferredexamples include phosphate groups and phosphorothioate groups.

The nucleotides of the antisense-oligonucleotides of the presentinvention or contiguous nucleotide sequences thereof are coupledtogether via internucleotide linkages. Suitably each nucleotide islinked through the 5′ position to the 3′ adjacent nucleotide via aninternucleotide linkage.

The antisense-oligonucleotides can be modified by several differentways. Modifications within the backbone are possible and refer toantisense-oligonucleotides wherein the phosphate groups (also namedphosphodiester groups) in their internucleotide backbone are partiallyor completely replaced by other groups. Preferred modifiedantisense-oligonucleotide backbones include, for instance,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriester, aminoalkylphosphotriesters, methyl, ethyl andC₃-C₁₀-alkyl phosphonates including 3′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogues ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleotide units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acids forms thereof are also included anddisclosed herein in further detail.

Suitable internucleotide linkages include those listed withinWO2007/031091, for example the internucleotide linkages listed on thefirst paragraph of page 34 of WO2007/031091 (hereby incorporated byreference). It is, in some embodiments, preferred to modify theinternucleotide linkage from its normal phosphodiester to one that ismore resistant to nuclease attack, such as phosphorothioate orboranophosphate—these two, accepted by RNase H mediated cleavage, alsoallow that route of antisense inhibition in reducing the expression ofthe target gene.

The internucleotide linkage consists of the group IL′ which is the groupbound to the 3′ carbon atom of the ribose moiety and the group Y whichis the group bound to the 5′ carbon atom of the contiguous ribose moietyas shown in the formula (IL′Y) below

The internucleotide linkage IL is represented by -IL′-Y—. IL′ represents—X″—P(═X′)(X⁻)— so that IL is represented by —X″—P(═X′)(X⁻)—Y—, whereinthe substituents X⁻, X′, X″ and Y have the meanings as disclosed herein.

The internucleotide linkage IL=—X″—P(═X′)(X⁻)—Y— is preferably selectedform the group consisting of:

—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—,—S—P(O)(S⁻)—O—, —S—P(S)(S⁻)—O—, —O—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—,—S—P(O)(O⁻)—S—, —O—P(O)(R^(H))—O—, —O—P(O)(OR^(H))—O—,—O—P(O)(NHR^(H))—O—, —O—P(O)[N(R^(H))₂]—O—, —O—P(O)(BH₃ ⁻)—O—,—O—P(O)(OCH₂CH₂OR^(H))—O—, —O—P(O)(OCH₂CH₂SR^(H))—O—,—O—P(O)(O⁻)—NR^(H)—, —NR^(H)—P(O)(O⁻)—O—, where R^(H) is selected fromhydrogen and C1-4-alkyl.

The group —O—P(O)(R^(H))—O— is preferably —O—P(O)(CH₃)—O— or—O—P(O)(C₂H₅)—O— and most preferably —O—P(O)(CH₃)—O—.

The group —O—P(O)(OR^(H))—O— is preferably —O—P(O)(OCH₃)—O— or—O—P(O)(OC₂H₅)—O— and most preferably —O—P(O)(OCH₃)—O—.

The group —O—P(O)(NHR^(H))—O— is preferably —O—P(O)(NHCH₃)—O— or—O—P(O)(NHC₂H₅)—O— and most preferably —O—P(O)(NHCH₃)—O—.

The group —O—P(O)[N(R^(H))₂]—O— is preferably —O—P(O)[N(CH₃)₂]—O— or—O—P(O)[N(C₂H₅)₂]—O— and most preferably —O—P(O)[N(CH₃)₂]—O—.

The group —O—P(O)(OCH₂CH₂OR^(H))—O— is preferably—O—P(O)(OCH₂CH₂OCH₃)—O— or —O—P(O)(OCH₂CH₂₀C₂H₅)—O— and most preferably—O—P(O)(OCH₂CH₂OCH₃)—O—.

The group —O—P(O)(OCH₂CH₂SR^(H))—O— is preferably—O—P(O)(OCH₂CH₂SCH₃)—O— or —O—P(O)(OCH₂CH₂SC₂H₅)—O— and most preferably—O—P(O)(OCH₂CH₂SCH₃)—O—.

The group —O—P(O)(O⁻)—NR^(H)— is preferably —O—P(O)(O⁻)—NH— or—O—P(O)(O⁻)—N(CH₃)— and most preferably —O—P(O)(O⁻)—NH—.

The group —NR^(H)—P(O)(O⁻)—O— is preferably —NH—P(O)(O⁻)—O— or—N(CH₃)—P(O)(O⁻)—O— and most preferably —NH—P(O)(O⁻)—O—.

Even more preferably IL represents —O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—,—O—P(S)(S⁻)—O—, —O—P(O)(NHR^(H))—O—, or —O—P(O)[N(R^(H))₂]—O—, and stillmore preferably IL represents —O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, or—O—P(S)(S⁻)—O—, and most preferably IL represents —O—P(O)(S⁻)—O—, or—O—P(O)(O⁻)—O—.

Thus IL is preferably a phosphate group (—O—P(O)(O⁻)—O—), aphosphorothioate group (—O—P(O)(S⁻)—O—) or a phosphorodithioate group(—O—P(S)(S⁻)—O—).

The nucleotide units or the nucleosides of theantisense-oligonucleotides are connected to each other byinternucleotide linkages so that within one antisense-oligonucleotidedifferent internucleotide linkages can be present. The LNA units arepreferably linked by internucleotide linkages which are not phosphategroups. The LNA units are linked to each other by a group IL which ispreferably selected from —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—,—O—P(O)(NHR^(H))—O—, and —O—P(O)[N(R^(H))₂]—O— and more preferably from—O—P(O)(S⁻)—O— and —O—P(S)(S⁻)—O—.

The non-LNA units are linked to each other by a group IL which ispreferably selected from —O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—,—O—P(O)(NHR^(H))—O—, and —O—P(O)[N(R^(H))₂]—O— and more preferably from—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O— and —O—P(S)(S⁻)—O—.

A non-LNA unit is linked to an LNA unit by a group IL which ispreferably selected from —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—,—O—P(O)(NHR^(H))—O—, and —O—P(O)[N(R^(H))₂]—O— and more preferably from—O—P(O)(S⁻)—O— and —O—P(S)(S⁻)—O—.

The term “LNA unit” as used herein refers to a nucleotide which islocked, i.e. to a nucleotide which has a bicyclic structure andespecially a bicyclic ribose structure and more especially a bicyclicribose structure as shown in general formula (II). The bridge “locks”the ribose in the 3′-endo (North) conformation. The ribose moiety of anLNA nucleotide is modified with an extra bridge connecting the 2′ oxygenand 4′ carbon. Alternatively used terms for LNA are bicyclic nucleotidesor bridged nucleotides, thus, an alternative term for LNA unit isbicyclic nucleotide unit or bridged nucleotide unit.

The term “non-LNA unit” as used herein refers to a nucleotide which isnot locked, i.e. to a nucleotide which has no bicyclic sugar moiety andespecially no bicyclic ribose structure and more especially no bicyclicribose structure as shown in general formula (II). The non-LNA units aremost preferably DNA units.

The term “DNA unit” as used herein refers to a nucleotide containing a2-deoxyribose as sugar. Thus, the nucleotide is made of a nucleobase anda 2-deoxyribose.

The term “unit” as used herein refers to a part or a fragment or amoiety of an antisense-oligonucleotide of the present invention. Thus a“unit” is not a complete molecule, it is a part or a fragment or amoiety of an antisense-oligonucleotide which has at least one positionfor a covalent linkage to another part or fragment or moiety of theantisense-oligonucleotide. For example, the general structures (B1) to(B6) are units, because they can be covalently linked through the groupY and IL′ or —O— and —O—P(O)(S⁻)— respectively. Preferably a unit is amoiety consisting of a pentose structure, a nucleobase connected to thepentose structure a 5′ radical group and an IL′ radical group.

The term “building block” or “monomer” as used herein refers to amolecule and especially to a nucleoside which is used in the synthesisof an antisense-oligonucleotide of the present invention. Examples arethe LNA molecules of general formula (I), wherein Y represents a5′-terminal group and IL′ represents a 3′-terminal group.

Suitable sulphur (S) containing internucleotide linkages as providedherein are preferred.

Furthermore, pure diastereomeric antisense-oligonucleotides arepreferred. Preferred are Sp- and Rp-diastereomers as shown below:

Preferred are phosphorothioate moieties in the backbone where at least50% of the internucleotide linkages are phosphorothioate groups. Alsopreferred is that the LNA units, if present, are linked throughphosphorothioates as internucleotide linkages. Most preferred is acomplete phosphorothioate backbone, i.e. most preferred is when allnucleotide units and also the LNA units (if present) are linked to eachother through phosphorothioate groups which are defined as follows:—O—P(O)(S⁻)—O-which is synonymous to —O—P(O,S)—O— or to —O—P(O—)(S)—O—.

In case the antisense-oligonucleotide is a gapmer, it is preferred thatthe LNA regions have internucleotide linkages selected from—O—P(O)(S⁻)—O— and —O—P(S)(S⁻)—O— and that the non-LNA region, themiddle part, has internucleotide linkages selected from —O—P(O)(O⁻)—O—,—O—P(O)(S⁻)—O— and —O—P(S)(S⁻)—O— and that the LNA regions are connectedto the non-LNA region through internucleotide linkages selected from—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O— and —O—P(S)(S⁻)—O—.

It is even more preferred if all internucleotide linkages which are 9 ina 10-mer and 19 in a 20-mer are selected from —O—P(O)(S⁻)—O— and—O—P(S)(S⁻)—O—. Still more preferred is that all internucleotidelinkages are phosphorothioate groups (—O—P(O)(S⁻)—O—) or arephosphorodithioate groups (—O—P(S)(S⁻)—O—).

Locked Nucleic Acids (LNA®)

It is especially preferred that some of the nucleotides of the generalformula (B1) or (B2) in the antisense-oligonucleotides are replaced byso-called LNAs (Locked Nucleic Acids). The abbreviation LNA is aregistered trademark, but herein the term “LNA” is solely used in adescriptive manner.

Preferably the terminal nucleotides are replaced by LNAs and morepreferred the last 1 to 4 nucleotides at the 3′ end and/or the last 1 to4 nucleotides at the 5′ end are replaced by LNAs. It is also preferredto have at least the terminal nucleotide at the 3′ end and at the 5′ endreplaced by an LNA each.

The term “LNA” as used herein, refers to a bicyclic nucleotide analogue,known as “Locked Nucleic Acid”. It may refer to an LNA monomer, or, whenused in the context of an “LNA antisense-oligonucleotide” or an“antisense-oligonucleotide containing LNAs”, LNA refers to anoligonucleotide containing one or more such bicyclic nucleotideanalogues. LNA nucleotides are characterized by the presence of a linkergroup (such as a bridge) between C₂′ and C₄′ of the ribose sugarring—for example as shown as the biradical R^(#)—R as described below.The LNA used in the antisense-oligonucleotides of the present inventionpreferably has the structure of the general formula (I)

wherein for all chiral centers, asymmetric groups may be found in eitherR or S orientation;wherein X is selected from —O—, —S—, —N(R^(N))—, —C(R⁶R⁷)—, andpreferably X is —O—;B is selected from hydrogen, optionally substituted C₁₋₄-alkoxy,optionally substituted C₁₋₄-alkyl, optionally substituted C₁₋₄-acyloxy,nucleobases and nucleobase analogues, and preferably B is a nucleobaseor a nucleobase analogue and most preferred a standard nucleobase;Y represents a part of an internucleotide linkage to an adjacentnucleotide in case the moiety of general formula (I) is an LNA unit ofan antisense-oligonucleotide of the present invention, or a 5′-terminalgroup in case the moiety of general formula (I) is a monomer or buildingblock for synthesizing an antisense-oligonucleotide of the presentinvention. The 5′ carbon atom optionally includes the substituent R⁴ andR⁵;IL′ represents a part of an internucleotide linkage to an adjacentnucleotide in case the moiety of general formula (I) is an LNA unit ofan antisense-oligonucleotide of the present invention, or a 3′-terminalgroup in case the moiety of general formula (I) is a monomer or buildingblock for synthesizing an antisense-oligonucleotide of the presentinvention.R^(#) and R together represent a bivalent linker group consisting of 1-4groups or atoms selected from —C(R^(a)R^(b))—, —C(R^(a))═C(R^(b))—,—C(R^(a))═N—, —O—, —Si(R^(a))₂—, —S—, —SO₂—, —N(R^(c))—, and >C═Z,wherein Z is selected from —O—, —S—, and —N(R^(a))—, and R^(a), R^(b)and R^(c) are independently of each other selected from hydrogen,optionally substituted C₁₋₁₂-alkyl, optionally substituted C₂₋₆-alkenyl,optionally substituted C₂₋₆-alkynyl, hydroxy, optionally substitutedC₁₋₁₂-alkoxy, C₁₋₆-alkoxy-C₁₋₆-alkyl, C₂₋₆-alkenyloxy, carboxy,C₁₋₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl, formyl, aryl,aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkylenyl-aminocarbonyl, mono-and di(C₁₋₆-alkyl)amino-C₁₋₆-alkylenyl-aminocarbonyl,C₁₋₆-alkyl-carbonylamino, carbamido, C₁₋₆-alkanoyloxy, sulphono,C₁₋₆-alkylsulphonyloxy, nitro, azido, sulphanyl, C₁₋₆-alkylthio,halogen, where aryl and heteroaryl may be optionally substituted andwhere two geminal substituents R^(a) and R^(b) together may representoptionally substituted methylene (═CH₂), wherein for all chiral centers,asymmetric groups may be found in either R or S orientation, and;each of the substituents R¹, R², R³, R⁴, R⁵, R⁶ and R⁷, which arepresent is independently selected from hydrogen, optionally substitutedC₁₋₁₂-alkyl, optionally substituted C₂₋₆-alkenyl, optionally substitutedC₂₋₆-alkynyl, hydroxy, C₁₋₁₂-alkoxy, C₁₋₆-alkoxy-C₁₋₆-alkyl,C₂₋₆-alkenyloxy, carboxy, C₁₋₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl,formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, where aryl and heteroaryl maybe optionally substituted, and where two geminal substituents togethermay designate oxo, thioxo, imino, or optionally substituted methylene;wherein R^(N) is selected from hydrogen and C₁₋₄-alkyl, and where twoadjacent (non-geminal) substituents may designate an additional bondresulting in a double bond; and R^(N), when present and not involved ina biradical, is selected from hydrogen and C₁₋₄-alkyl; and basic saltsand acid addition salts thereof. For all chiral centers, asymmetricgroups may be found in either R or S orientation.

In preferred embodiments, R^(#) and R together represent a biradicalconsisting of a groups selected from the group consisting of—C(R^(a)R^(b))—C(R^(a)R^(b))—, —C(R^(a)R^(b))—O—,—C(R^(a)R^(b))—NR^(c)—, —C(R^(a)R^(b))—S—, and—C(R^(a)R^(b))—C(R^(a)R^(b))—O—, wherein each R^(a), R^(b) and R^(c) mayoptionally be independently selected.

In some embodiments, R^(a) and R^(b) may be, optionally independentlyselected from the group consisting of hydrogen and C₁₋₆-alkyl, such asmethyl, and preferred is hydrogen.

In preferred embodiments, R¹, R², R³, R⁴, and R⁵ are independentlyselected from the group consisting of hydrogen, halogen, C₁₋₆-alkyl,substituted C₁₋₆-alkyl, C₂₋₆-alkenyl, substituted C₂₋₆-alkenyl,C₂₋₆-alkynyl or substituted C₂₋₆-alkynyl, C₁₋₆-alkoxyl, substitutedC₁₋₆-alkoxyl, acyl, substituted acyl, C₁₋₆-aminoalkyl or substitutedC₁₋₆-aminoalkyl. For all chiral centers, asymmetric groups may be foundin either R or S orientation.

In preferred embodiments R¹, R², R³, R⁴, and R⁵ are hydrogen.

In some embodiments, R¹, R², and R³, are independently selected from thegroup consisting of hydrogen, halogen, C₁₋₆-alkyl, substitutedC₁₋₆-alkyl, C₂₋₆-alkenyl, substituted C₂₋₆-alkenyl, C₂₋₆-alkynyl orsubstituted C₂₋₆-alkynyl, C₁₋₆-alkoxyl, substituted C₁₋₆-alkoxyl, acyl,substituted acyl, C₁₋₆-aminoalkyl or substituted C₁₋₆-aminoalkyl. Forall chiral centers, asymmetric groups may be found in either R or Sorientation. In preferred embodiments R¹, R², and R³ are hydrogen.

In preferred embodiments, R⁴ and R⁵ are each independently selected fromthe group consisting of —H, —CH₃, —CH₂—CH₃, —CH₂—O—CH₃, and —CH═CH₂.Suitably in some embodiments, either R⁴ or R⁵ are hydrogen, whereas theother group (R⁴ or R⁵ respectively) is selected from the groupconsisting of C₁₋₆-alkyl, C₂₋₆-alkenyl, C₂₋₆-alkynyl, substitutedC₁₋₆-alkyl, substituted C₂₋₆-alkenyl, substituted C₂₋₆-alkynyl orsubstituted acyl (—C(═O)—); wherein each substituted group is mono orpoly substituted with substituent groups independently selected fromhalogen, C₁₋₆-alkyl, substituted C₁₋₆-alkyl, C₂₋₆-alkenyl, substitutedC₂₋₆-alkenyl, C₂₋₆-alkynyl, substituted C₂₋₆-alkynyl, -OJ₁, -SJ₁,-NJ₁J₂, —N₃, -COOJ₁, —CN, —O—C(═O)NJ₁J₂, —N(H)C(═NH)NJ₁J₂ or—N(H)C(═X)N(H)J₂, wherein X is O or S; and each J₁ and J₂ is,independently —H, C₁₋₆-alkyl, substituted C₁₋₆-alkyl, C₂₋₆-alkenyl,substituted C₂₋₆-alkenyl, C₂₋₆-alkynyl, substituted C₂₋₆-alkynyl,C₁₋₆-aminoalkyl, substituted C₁₋₆-aminoalkyl or a protecting group. Insome embodiments either R⁴ or R⁵ is substituted C₁₋₆-alkyl. In someembodiments either R⁴ or R⁵ is substituted methylene, wherein preferredsubstituent groups include one or more groups independently selectedfrom —F, -NJ₁J₂, —N₃, —CN, -OJ₁, -SJ₁, —O—C(═O)NJ₁J₂, —N(H)C(═NH)NJ₁J₂or —N(H)C(═O)N(H)J₂. In some embodiments each J₁ and J₂ is,independently —H or C₁₋₆-alkyl. In some embodiments either R⁴ or R⁵ ismethyl, ethyl or methoxymethyl. In some embodiments either R⁴ or R⁵ ismethyl. In a further embodiment either R⁴ or R⁵ is ethylenyl. In someembodiments either R⁴ or R⁵ is substituted acyl. In some embodimentseither R⁴ or R⁵ is —O—C(═O)NJ₁J₂. For all chiral centers, asymmetricgroups may be found in either R or S orientation. Such 5′ modifiedbicyclic nucleotides are disclosed in WO 2007/134181 A, which is herebyincorporated by reference in its entirety.

In some embodiments B is a nucleobase, including nucleobase analoguesand naturally occurring nucleobases, such as a purine or pyrimidine, ora substituted purine or substituted pyrimidine, such as a nucleobasereferred to herein, such as a nucleobase selected from the groupconsisting of adenine, cytosine, thymine, adenine, uracil, and/or amodified or substituted nucleobase, such as 5-thiazolo-uracil,2-thio-uracil, 5-propynyl-uracil, 2′thio-thymine, 5-methyl cytosine,5-thiozolo-cytosine, 5-propynyl-cytosine, and 2,6- diaminopurine.

In preferred embodiments, R^(#) and R together represent a biradicalselected from —C(R^(a)R^(b))—O—, —C(R^(a)R^(b))—C(R^(c)R^(d))—O—,—C(R^(a)R^(b))—C(R^(c)R^(d))—C(R^(e)R^(f))—O—,—C(R^(a)R^(b))—O—C(R^(d)R^(e))—, —C(R^(a)R^(b))—O—C(R^(d)R^(e))—O—,—C(R^(a)R^(b))—C(R^(d)R^(e))—,—C(R^(a)R^(b))—C(R^(c)R^(d))—C(R^(e)R^(f))—,—C(R^(a))═C(R^(b))—C(R^(d)R^(e))—, —C(R^(a)R^(b))—N(R^(c))—,—C(R^(a)R^(b))—C(R^(d)R^(e))—N(R^(c))—, —C(R^(a)R^(b))—N(R^(c))—O—,—C(R^(a)R^(b))—S—, and —C(R^(a)R^(b))—C(R^(d)R^(e))—S—, wherein R^(a),R^(b), R^(c), R^(d), R^(e), and R^(f) each is independently selectedfrom hydrogen, optionally substituted C₁₋₁₂-alkyl, optionallysubstituted C₂₋₆-alkenyl, optionally substituted C₂₋₆-alkynyl, hydroxy,C₁₋₁₂-alkoxy, C₁₋₆-alkoxy-C₁₋₆-alkyl, C₂₋₆-alkenyloxy, carboxy,C₁₋₁₂-alkoxycarbonyl C₁₋₁₂-alkylcarbonyl, formyl, aryl,aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, where aryl and heteroaryl maybe optionally substituted and where two geminal substituents R^(a) andR^(b) together may designate optionally substituted methylene (═CH₂).For all chiral centers, asymmetric groups may be found in either R or Sorientation.

In a further embodiment R^(#) and R together designate a biradical(bivalent group) selected from —CH₂—O—, —CH₂—S—, —CH₂—NH—, —CH₂—N(CH₃)—,—CH₂—CH₂—O—, —CH₂—CH(CH₃)—, —CH₂—CH₂—S—, —CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—,—CH₂—CH₂—CH₂—O—, —CH₂—CH₂—CH(CH₃)—, —CH═CH—CH₂—, —CH₂—O—CH₂—O—,—CH₂—NH—O—, —CH₂—N(CH₃)—O—, —CH₂—O—CH₂—, —CH(CH₃)—O—, —CH(CH₂—O—CH₃)—O—,—CH₂—CH₂—, and —CH═CH—. For all chiral centers, asymmetric groups may befound in either R or S orientation.

In some embodiments, R^(#) and R together designate the biradical—C(R^(a)R^(b))—N(R^(c))—O—, wherein R^(a) and R^(b) are independentlyselected from the group consisting of hydrogen, halogen, C₁₋₆-alkyl,substituted C₁₋₆-alkyl, C₂₋₆-alkenyl, substituted C₂₋₆-alkenyl,C₂₋₆-alkynyl or substituted C₂₋₆-alkynyl, C₁₋₆-alkoxyl, substitutedC₁₋₆-alkoxyl, acyl, substituted acyl, C₁₋₆-aminoalkyl or substitutedC₁₋₆-aminoalkyl, such as hydrogen, and; wherein R^(c) is selected fromthe group consisting of hydrogen, halogen, C₁₋₆-alkyl, substitutedC₁₋₆-alkyl, C₂₋₆-alkenyl, substituted C₂₋₆-alkenyl, C₂₋₆-alkynyl orsubstituted C₂₋₆-alkynyl, C₁₋₆-alkoxyl, substituted C₁₋₆-alkoxyl, acyl,substituted acyl, C₁₋₆-aminoalkyl or substituted C₁₋₆-aminoalkyl, andpreferably hydrogen.

In preferred embodiments, R^(#) and R together represent the biradical—C(R^(a)R^(b))—O—C(R^(d)R^(e))—O—, wherein R^(a), R^(b), R^(d), andR^(e) are independently selected from the group consisting of hydrogen,halogen, C₁₋₆-alkyl, substituted C₁₋₆-alkyl, C₂₋₆-alkenyl, substitutedC₂₋₆-alkenyl, C₂₋₆-alkynyl or substituted C₂₋₆-alkynyl, C₁₋₆-alkoxyl,substituted C₁₋₆-alkoxyl, acyl, substituted acyl, C₁₋₆-aminoalkyl orsubstituted C₁₋₆-aminoalkyl, and preferably hydrogen.

In preferred embodiments, R^(#) and R form the biradical —CH(Z)—O—,wherein Z is selected from the group consisting of C₁₋₆-alkyl,C₂₋₆-alkenyl, C₂₋₆-alkynyl, substituted C₁₋₆-alkyl, substitutedC₂₋₆-alkenyl, substituted C₂₋₆-alkynyl, acyl, substituted acyl,substituted amide, thiol or substituted thio; and wherein each of thesubstituted groups, is, independently, mono or poly substituted withoptionally protected substituent groups independently selected fromhalogen, oxo, hydroxyl, -OJ₁, —NJ₁J₂, -SJ₁, —N₃, —OC(═X)J₁,—OC(═X)NJ₁J₂, -NJ³C(═X)NJ₁J₂ and —CN, wherein each J₁, J₂ and J₃ is,independently, —H or C₁₋₆-alkyl, and X is O, S or NJ₁. In preferredembodiments Z is C₁₋₆-alkyl or substituted C₁₋₆-alkyl. In furtherpreferred embodiments Z is methyl. In preferred embodiments Z issubstituted C₁₋₆-alkyl. In preferred embodiments said substituent groupis C₁₋₆-alkoxy. In some embodiments Z is CH₃OCH₂—. For all chiralcenters, asymmetric groups may be found in either R or S orientation.Such bicyclic nucleotides are disclosed in U.S. Pat. No. 7,399,845 whichis hereby incorporated by reference in its entirety. In preferredembodiments, R¹, R², R³, R⁴, and R⁵ are hydrogen. In preferredembodiments, R¹, R², and R³ are hydrogen, and one or both of R⁴, R⁵ maybe other than hydrogen as referred to above and in WO 2007/134181.

In preferred embodiments, R^(#) and R together represent a biradicalwhich comprise a substituted amino group in the bridge such as thebiradical —CH₂—N(R^(c))—, wherein R^(c) is C₁₋₁₂-alkyloxy. In preferredembodiments R^(#) and R together represent a biradical -Cq₃q₄-NOR—,wherein q₃ and q₄ are independently selected from the group consistingof hydrogen, halogen, C₁₋₆-alkyl, substituted C₁₋₆-alkyl, C₂₋₆-alkenyl,substituted C₂₋₆-alkenyl, C₂₋₆-alkynyl or substituted C₂₋₆-alkynyl,C₁₋₆-alkoxyl, substituted C₁₋₆-alkoxyl, acyl, substituted acyl,C₁₋₆-aminoalkyl or substituted C₁₋₆-aminoalkyl; wherein each substitutedgroup is, independently, mono or poly substituted with substituentgroups independently selected from halogen, -OJ₁, —SJ₁, -NJ₁J₂, -COOJ₁,—CN, —OC(═O)NJ₁J₂, —NH—C(═NH)NJ₁J₂ or —NH—C(═X)NHJ₂, wherein X is O orS; and each of J₁ and J₂ is, independently, —H, C₁₋₆-alkyl,C₂₋₆-alkenyl, C₂₋₆-alkynyl, C₁₋₆-aminoalkyl or a protecting group. Forall chiral centers, asymmetric groups may be found in either R or Sorientation. Such bicyclic nucleotides are disclosed in WO2008/150729which is hereby incorporated by reference in its entirety. In preferredembodiments, R¹, R², R³, R⁴, and R⁵ are independently selected from thegroup consisting of hydrogen, halogen, C₁₋₆-alkyl, substitutedC₁₋₆-alkyl, C₂₋₆-alkenyl, substituted C₂₋₆-alkenyl, C₂₋₆-alkynyl orsubstituted C₂₋₆-alkynyl, C₁₋₆-alkoxyl, substituted C₁₋₆-alkoxyl, acyl,substituted acyl, C₁₋₆-aminoalkyl or substituted C₁₋₆-aminoalkyl. Inpreferred embodiments, R¹, R², R³, R⁴, and R⁵ are hydrogen. In preferredembodiments, R¹, R², and R³ are hydrogen and one or both of R⁴, R⁵ maybe other than hydrogen as referred to above and in WO 2007/134181.

In preferred embodiments R^(#) and R together represent a biradical(bivalent group) —C(R^(a)R^(b))—O—, wherein R^(a) and R^(b) are eachindependently halogen, C₁₋₁₂-alkyl, substituted C₁₋₁₂-alkyl,C₂₋₆-alkenyl, substituted C₂₋₆-alkenyl, C₂₋₆-alkynyl, substitutedC₂₋₆-alkynyl, C₁₋₁₂-alkoxy, substituted C₁₋₁₂-alkoxy, -OJ₁, -SJ₁,—S(O)J₁, —SO₂-J₁, —NJ₁J₂, —N₃, —CN, —C(═O)OJ₁, —C(═O)NJ₁J₂, —C(═O)J₁,—OC(═O)NJ₁J₂, —NH—C(═NH)NJ₁J₂, —NH—C(═O)NJ₁J₂, or, —NH—C(═S)NJ₁J₂; orR^(a) and R^(b) together are ═C(q₃)(q₄); q₃ and q₄ are each,independently, —H, halogen, C₁₋₁₂-alkyl or substituted C₁₋₁₂-alkyl; eachsubstituted group is, independently, mono or poly substituted withsubstituent groups independently selected from halogen, C₁₋₆-alkyl,substituted C₁₋₆-alkyl, C₂₋₆-alkenyl, substituted C₂₋₆-alkenyl,C₂₋₆-alkynyl, substituted C₂₋₆-alkynyl, -OJ₁, -SJ₁, -NJ₁J₂, —N₃, —CN,—C(═O)OJ₁, —C(═O)NJ₁J₂, —C(═O)J₁, —OC(═O)NJ₁J₂, —NH—C(═O)NJ₁J₂, or—NH—C(═S)NJ₁J₂ and; each J₁ and J₂ is independently, —H, C₁₋₆-alkyl,substituted C₁₋₆-alkyl, C₂₋₆-alkenyl, substituted C₂₋₆-alkenyl,C₂₋₆-alkynyl, substituted C₂₋₆-alkynyl, C₁₋₆-aminoalkyl, substitutedC₁₋₆-aminoalkyl or a protecting group. Such compounds are disclosed inWO2009006478A, hereby incorporated in its entirety by reference.

In preferred embodiments, R^(#) and R form the biradical -Q-, wherein Qis —C(q₁)(q₂)C(q₃)(q₄)-, —C(q₁)═C(q₃)-, —C[═C(q₁)(q₂)]—C(q₃)(q₄)- or—C(q₁)(q₂)—C[═C(q₃)(q₄)]-;

q₁, q₂, q₃, q₄ are each independently of each other —H, halogen,C₁₋₁₂-alkyl, substituted C₁₋₁₂-alkyl, C₂₋₆-alkenyl, substitutedC₁₋₁₂-alkoxy, -OJ₁, -SJ₁, —S(O)J₁, —SO₂-J₁, -NJ₁J₂, —N₃, —CN, —C(═O)OJ₁,—C(═O)NJ₁J₂, —C(═O)J₁, —OC(═O)NJ₁J₂, —NH—C(═NH)NJ₁J₂, —NH—C(═O)NJ₁J₂, or—NH—C(═S)NJ₁J₂; each J₁ and J₂ is independently of each other —H,C₁₋₆-alkyl, C₂₋₆-alkenyl, C₂₋₆-alkynyl, C₁₋₆-aminoalkyl or a protectinggroup; and optionally when Q is —C(q₁)(q₂)C(q₃)(q₄)- and one of q₃ or q₄is —CH₃, then at least one of the other of q₃ or q₄ or one of q₁ and q₂is other than —H. In preferred embodiments R¹, R², R³, R⁴, and R⁵ arehydrogen. For all chiral centers, asymmetric groups may be found ineither R or S orientation. Such bicyclic nucleotides are disclosed inWO2008/154401 which is hereby incorporated by reference in its entirety.In preferred embodiments R¹, R², R³, R⁴, and R⁵ are independently ofeach other selected from the group consisting of hydrogen, halogen,C₁₋₆-alkyl, substituted C₁₋₆-alkyl, C₂₋₆-alkenyl, substitutedC₂₋₆-alkenyl, C₂₋₆-alkynyl or substituted C₂₋₆-alkynyl, C₁₋₆-alkoxyl,substituted C₁₋₆-alkoxyl, acyl, substituted acyl, C₁₋₆-aminoalkyl orsubstituted C₁₋₆-aminoalkyl. In preferred embodiments R¹, R², R³, R⁴,and R⁵ are hydrogen. In preferred embodiments R¹, R², and R³ arehydrogen and one or both of R⁴, R⁵ may be other than hydrogen asreferred to above and in WO 2007/134181 or WO2009/067647(alpha-L-bicyclic nucleic acids analogues).

As used herein, the term “C₁-C₆-alkyl” refers to —CH₃, —C₂H₅, —C₃H₇,—CH(CH₃)₂, —C₄H₉, —CH₂—CH(CH₃)₂, —CH(CH₃)—C₂H₅, —C(CH₃)₃, —C₅H₁₁,—CH(CH₃)—C₃H₇, —CH₂—CH(CH₃)—C₂H₅, —CH(CH₃)—CH(CH₃)₂, —C(CH₃)₂—C₂H₅,—CH₂—C(CH₃)₃, —CH(C₂H₅)₂, —C₂H₄—CH(CH₃)₂, —C₆H₁₃, —C₃H₆—CH(CH₃)₂,—C₂H₄—CH(CH₃)—C₂H₅, —CH(CH₃)—C₄H₉, —CH₂—CH(CH₃)—C₃H₇,—CH(CH₃)—CH₂—CH(CH₃)₂, —CH(CH₃)—CH(CH₃)—C₂H₅, —CH₂—CH(CH₃)—CH(CH₃)₂,—CH₂—C(CH₃)₂—C₂H₅, —C(CH₃)₂—C₃H₇, —C(CH₃)₂—CH(CH₃)₂, —C₂H₄—C(CH₃)₃,—CH₂—CH(C₂H₅)₂, and —CH(CH₃)—C(CH₃)₃. The term “C₁-C₆-alkyl” shall alsoinclude “C₁-C₆-cycloalkyl” like cyclo-C₃H₅, cyclo-C₄H₇, cyclo-C₅H₉, andcyclo-C₆H₁₁.

Preferred are —CH₃, —C₂H₅, —C₃H₇, —CH(CH₃)₂, —C₄H₉, —CH₂—CH(CH₃)₂,—CH(CH₃)—C₂H₅, —C(CH₃)₃, and —C₅H₁₁. Especially preferred are —CH₃,—C₂H₅, —C₃H₇, and —CH(CH₃)₂.

The term “C₁-C₆-alkyl” shall also include “C₁-C₆-cycloalkyl” likecyclo-C₃H₅, cyclo-C₄H₇, cyclo-C₅H₉, and cyclo-C₆H₁₁.

As used herein, the term “C₁-C₁₂-alkyl” refers to C₁-C₆-alkyl, —C₇H₁₅,—C₈H₁₇, —C₉H₁₉, —C₁₀H₂₁, —C₁₁H₂₃, —C₁₂H₂₅.

As used herein, the term “C₁-C₆-alkylenyl” refers to —CH₂—, —C₂H₄—,—CH(CH₃)—, —C₃H₆—, —CH₂—CH(CH₃)—, —CH(CH₃)—CH₂—, —C(CH₃)₂—, —C₄H₈—,—CH₂—C(CH₃)₂—, —C(CH₃)₂—CH₂—, —C₂H₄—CH(CH₃)—, —CH(CH₃)—C₂H₄—,—CH₂—CH(CH₃)—CH₂—, —CH(CH₃)—CH(CH₃)—, —C₅H₁₀—, —CH(CH₃)—C₃H₆—,—CH₂—CH(CH₃)—C₂H₄—, —C₂H₄—CH(CH₃)—CH₂—, —C₃H₆—CH(CH₃)—, —C₂H₄—C(CH₃)₂—,—C(CH₃)₂—C₂H₄—, —CH₂—C(CH₃)₂—CH₂—, —CH₂—CH(CH₃)—CH(CH₃)—,—CH(CH₃)—CH₂—CH(CH₃)—, —CH(CH₃)—CH(CH₃)—CH₂—, —CH(CH₃)—CH(CH₃)—CH(CH₃)—,—C(CH₃)₂—C₃H₆—, —CH₂—C(CH₃)₂—C₂H₄—, —C₂H₄—C(CH₃)₂—CH₂—, —C₃H₆—C(CH₃)₂—,—CH(CH₃)—C₄H₈—, —C₆H₁₂—, —CH₂—CH(CH₃)—C₃H₆—, —C₂H₄—CH(CH₃)—C₂H₄—,—C₃H₆—CH(CH₃)—CH₂—, —C₄H₈—CH(CH₃)—, —C₂H₄—CH(CH₃)—CH(CH₃)—,—CH₂—CH(CH₃)—CH(CH₃)—CH₂—, —CH₂—CH(CH₃)—CH₂—CH(CH₃)—,—CH(CH₃)—C₂H₄—CH(CH₃)—, —CH(CH₃)—CH₂—CH(CH₃)—CH₂—, and—CH(CH₃)—CH(CH₃)—C₂H₄—.

As used herein, the term “C₂-C₆-alkenyl” refers to —CH═CH₂, —CH₂—CH═CH₂,—C(CH₃)═CH₂, —CH═CH—CH₃, —C₂H₄—CH═CH₂, —CH₂—CH═CH—CH₃, —CH═CH—C₂H₅,—CH₂—C(CH₃)═CH₂, —CH(CH₃)—CH═CH, —CH═C(CH₃)₂, —C(CH₃)═CH—CH₃,—CH═CH—CH═CH₂, —C₃H₆—CH═CH₂, —C₂H₄—CH═CH—CH₃, —CH₂—CH═CH—C₂H₅,—CH═CH—C₃H₇, —CH₂—CH═CH—CH═CH₂, —CH═CH—CH═CH—CH₃, —CH═CH—CH₂—CH═CH₂,—C(CH₃)═CH—CH═CH₂, —CH═C(CH₃)—CH═CH₂, —CH═CH—C(CH₃)═CH₂,—C₂H₄—C(CH₃)═CH₂, —CH₂—CH(CH₃)—CH═CH₂, —CH(CH₃)—CH₂—CH═CH₂,—CH₂—CH═C(CH₃)₂, —CH₂—C(CH₃)═CH—CH₃, —CH(CH₃)—CH═CH—CH₃,—CH═CH—CH(CH₃)₂, —CH═C(CH₃)—C₂H₅, —C(CH₃)═CH—C₂H₅, —C(CH₃)═C(CH₃)₂,—C(CH₃)₂—CH═CH₂, —CH(CH₃)—C(CH₃)═CH₂, —C(CH₃)═CH—CH═CH₂,—CH═C(CH₃)—CH═CH₂, —CH═CH—C(CH₃)═CH₂, —C₄H₈—CH═CH₂, —C₃H₆—CH═CH—CH₃,—C₂H₄—CH═CH—C₂H₅, —CH₂—CH═CH—C₃H₇, —CH═CH—C₄H₉, —C₃H₆—C(CH₃)═CH₂,—C₂H₄—CH(CH₃)—CH═CH₂, —CH₂—CH(CH₃)—CH₂—CH═CH₂, —CH(CH₃)—C₂H₄—CH═CH₂,—C₂H₄—CH═C(CH₃)₂, —C₂H₄—C(CH₃)═CH—CH₃, —CH₂—CH(CH₃)—CH═CH—CH₃,—CH(CH₃)—CH₂—CH═CH—CH₃, —CH₂—CH═CH—CH(CH₃)₂, —CH₂—CH═C(CH₃)—C₂H₅,—CH₂—C(CH₃)═CH—C₂H₅, —CH(CH₃)—CH═CH—C₂H₅, —CH═CH—CH₂—CH(CH₃)₂,—CH═CH—CH(CH₃)—C₂H₅, —CH═C(CH₃)—C₃H₇, —C(CH₃)═CH—C₃H₇,—CH₂—CH(CH₃)—C(CH₃)═CH₂, —CH(CH₃)—CH₂—C(CH₃)═CH₂,—CH(CH₃)—CH(CH₃)—CH═CH₂, —CH₂—C(CH₃)₂—CH═CH₂, —C(CH₃)₂—CH₂—CH═CH₂,—CH₂—C(CH₃)═C(CH₃)₂, —CH(CH₃)—CH═C(CH₃)₂, —C(CH₃)₂—CH═CH—CH₃,—CH(CH₃)—C(CH₃)═CH—CH₃, —CH═C(CH₃)—CH(CH₃)₂, —C(CH₃)═CH—CH(CH₃)₂,—C(CH₃)═C(CH₃)—C₂H₅, —CH═CH—C(CH₃)₃, —C(CH₃)₂—C(CH₃)═CH₂,—CH(C₂H₅)—C(CH₃)═CH₂, —C(CH₃)(C₂H₅)—CH═CH₂, —CH(CH₃)—C(C₂H₅)═CH₂,—CH₂—C(C₃H₇)═CH₂, —CH₂—C(C₂H₅)═CH—CH₃, —CH(C₂H₅)—CH═CH—CH₃,—C(C₄H₉)═CH₂, —C(C₃H₇)═CH—CH₃, —C(C₂H₅)═CH—C₂H₅, —C(C₂H₅)═C(CH₃)₂,—C[C(CH₃)₃]═CH₂, —C[CH(CH₃)(C₂H₅)]═CH₂, —C[CH₂—CH(CH₃)₂]═CH₂,—C₂H₄—CH═CH—CH═CH₂, —CH₂—CH═CH—CH₂—CH═CH₂, —CH═CH—C₂H₄—CH═CH₂,—CH₂—CH═CH—CH═CH—CH₃, —CH═CH—CH₂—CH═CH—CH₃, —CH═CH—CH═CH—C₂H₅,—CH₂—CH═CH—C(CH₃)═CH₂, —CH₂—CH═C(CH₃)—CH═CH₂, —CH₂—C(CH₃)═CH—CH═CH₂,—CH(CH₃)—CH═CH—CH═CH₂, —CH═CH—CH₂—C(CH₃)═CH₂, —CH═CH—CH(CH₃)—CH═CH₂,—CH═C(CH₃)—CH₂—CH═CH₂, —C(CH₃)═CH—CH₂—CH═CH₂, —CH═CH—CH═C(CH₃)₂,—CH═CH—C(CH₃)═CH—CH₃, —CH═C(CH₃)—CH═CH—CH₃, —C(CH₃)═CH—CH═CH—CH₃,—CH═C(CH₃)—C(CH₃)═CH₂, —C(CH₃)═CH—C(CH₃)═CH₂, —C(CH₃)═C(CH₃)—CH═CH₂, and—CH═CH—CH═CH—CH═CH₂.

Preferred are —CH═CH₂, —CH₂—CH═CH₂, —C(CH₃)═CH₂, —CH═CH—CH₃,—C₂H₄—CH═CH₂, —CH₂—CH═CH—CH₃. Especially preferred are —CH═CH₂,—CH₂—CH═CH₂, and —CH═CH—CH₃.

As used herein, the term “C₂-C₆-alkynyl” refers to —C≡CH, —C≡C—CH₃,—CH₂—C≡CH, —C₂H₄—C≡CH, —CH₂—C≡C—CH₃, —C≡C—C₂H₅, —C₃H₆—C≡CH,—C₂H₄—C≡C—CH₃, —CH₂—C≡C₂H₅, —C≡C—C₃H₇, —CH(CH₃)—C≡CH, —CH₂—CH(CH₃)—C≡CH,—CH(CH₃)—CH₂—C≡CH, —CH(CH₃)—C≡C—CH₃, —C₄H₈—C≡CH, —C₃H₆—C≡C—CH₃,—C₂H₄—C≡C—C₂H₅, —CH₂—C≡C—C₃H₇, —C≡C—C₄H₉, —C₂H₄—CH(CH₃)—C≡CH,—CH₂—CH(CH₃)—CH₂—C≡CH, —CH(CH₃)—C₂H₄—C≡CH, —CH₂—CH(CH₃)—C≡C—CH₃,—CH(CH₃)—CH₂—C≡C—CH₃, —CH(CH₃)—C≡C—C₂H₅, —CH₂—C≡C—CH(CH₃)₂,—C≡C—CH(CH₃)—C₂H₅, —C≡C—CH₂—CH(CH₃)₂, —C≡C—C(CH₃)₃, —CH(C₂H₅)—C≡C—CH₃,—C(CH₃)₂—C≡C—CH₃, —CH(C₂H₅)—CH₂—C≡CH, —CH₂—CH(C₂H₅)—C≡CH,—C(CH₃)₂—CH₂—C≡CH, —CH₂—C(CH₃)₂—C≡CH, —CH(CH₃)—CH(CH₃)—C≡CH,—CH(C₃H₇)—C≡CH, —C(CH₃)(C₂H₅)—C≡CH, —C≡C—C≡CH, —CH₂—C≡C—C≡CH,—C—C—C≡C—CH₃, —CH(C—CH)₂, —C₂H₄—C≡C—C≡CH, —CH₂—C≡C—CH₂—C≡CH,—C≡C—C₂H₄—C≡CH, —CH₂—C≡C—C≡C—CH₃, —C≡C—CH₂—C≡C—CH₃, —C≡C—C≡C—C₂H₅,—C≡C—CH(CH₃)—C≡CH, —CH(CH₃)—C≡C—C≡CH, —CH(C≡CH)—CH₂—C≡CH, —C(C≡CH)₂—CH₃,—CH₂—CH(C≡CH)₂, —CH(C≡CH)—C≡C—CH₃. Preferred are —C≡CH and —C≡C—CH₃.

The term “C₁₋₆-alkoxyl” refers to “C₁-C₆-alkyl-O—”.

The term “C₁₋₁₂-alkoxyl” refers to “C₁-C₁₂-alkyl-O—”.

The term “C₁₋₆-aminoalkyl” refers to “H₂N—C₁-C₆-alkyl-”.

The term “C₂-C₆-alkenyloxy” refers to “C₂-C₆-alkenyl-O—”.

The term “C₁₋₆-alkylcarbonyl” refers to “C₁-C₆-alkyl-CO—”. Also referredto as “acyl”.

The term “C₁₋₁₂-alkylcarbonyl” refers to “C₁-C₁₂-alkyl-CO—”. Alsoreferred to as “acyl”.

The term “C₁₋₆-alkoxycarbonyl” refers to “C₁-C₆-alkyl-O—CO—”.

The term “C₁₋₁₂-alkoxycarbonyl” refers to “C₁-C₁₂-alkyl-O—CO—”.

The term “C₁-C₆-alkanoyloxy” refers to “C₁-C₆-alkyl-CO—O—”.

The term “C₁₋₆-alkylthio” refers to “C₁-C₆-alkyl-S—”.

The term “C₁₋₆-alkylsulphonyloxy” refers to “C₁-C₆-alkyl-SO₂—O—”.

The term “C₁₋₆-alkylcarbonylamino” refers to “C₁-C₆-alkyl-CO—NH—”.

The term “C₁₋₆-alkylamino” refers to “C₁-C₆-alkyl-NH—”.

The term “(C₁₋₆-)₂alkylamino” refers to a dialkylamino group like“[C₁-C₆-alkyl][C₁-C₆-alkyl]N—”.

The term “C₁₋₆-alkylaminocarbonyl” refers to “C₁-C₆-alkyl-NH—CO—”

The term “(C₁₋₆-)₂alkylaminocarbonyl” refers to a dialkylaminocarbonylgroup like “[C₁-C₆-alkyl][C₁-C₆-alkyl]N—CO—”.

The term “amino-C₁₋₆-alkylaminocarbonyl” refers to“H₂N—[C₁-C₆-alkylenyl]-N H—CO—”.

The term “C₁₋₆-alkyl-amino-C₁₋₆-alkylaminocarbonyl” refers to“C₁₋₆-alkyl-H N—[C₁-C₆-alkylenyl]-N H—CO—”.

The term “(C₁₋₆-)₂alkyl-amino-C₁₋₆-alkylaminocarbonyl” refers to“[C₁-C₆-alkyl][C₁-C₆-alkyl]N—[C₁-C₆-alkylenyl]-N H—CO—”.

The term “aryl” refers to phenyl, toluyl, substituted phenyl andsubstituted toluyl.

The term “aryloxy” refers to “aryl-O—”.

The term “arylcarbonyl” refers to “aryl-CO—”.

The term “aryloxycarbonyl” refers to “aryl-O—CO—”.

The term “heteroaryl” refers to substituted or not substitutedheteroaromatic groups which have from 4 to 9 ring atoms, from 1 to 4 ofwhich are selected from O, N and/or S. Preferred “heteroaryl” groupshave 1 or 2 heteroatoms in a 5- or 6-membered aromatic ring. Mono andbicyclic ring systems are included. Typical “heteroaryl” groups arepyridyl, furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl,isoxazolyl, isothiazolyl, oxadiazolyl, pyridazinyl, pyrimidyl,pyrazinyl, 1,3,5-triazinyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl,indolizinyl, indolyl, isoindolyl, benzo[b]furyl, benzo[b]thienyl,indazolyl, benzimidazolyl, benzthiazolyl, purinyl, quinolizinyl,quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, 1,8-naphthyridinyl,tetrahydroquinolyl, benzooxazolyl, chrom-2-onyl, indazolyl, and thelike.

The term “heteroaryloxy” refers to “heteroaryl-O—”.

The term “heteroarylcarbonyl” refers to “heteroaryl-CO—”.

The term “heteroaryloxycarbonyl” refers to “heteroaryl-O—CO—”.

The term “substituted” refers to groups wherein one or more hydrogenatoms are replaced by one or more of the following substituents: —OH,—OCH₃, —OC₂H₅, —OC₃H₇, —O-cyclo-C₃H₅, —OCH(CH₃)₂, —OCH₂Ph, —F, —Cl,—COCH₃, —COC₂H₅, —COC₃H₇, —CO-cyclo-C₃H₅, —COCH(CH₃)₂, —COOH, —CONH₂,—NH₂, —NHCH₃, —NHC₂H₅, —NHC₃H₇, —NH-cyclo-C₃H₅, —NHCH(CH₃)₂, —N(CH₃)₂,—N(C₂H₅)₂, —N(C₃H₇)₂, —N(cyclo-C₃H₅)₂, —N[CH(CH₃)₂]₂, —SO₃H, —OCF₃,—OC₂F₅, cyclo-C₃H₅, —CH₃, —C₂H₅, —C₃H₇, —CH(CH₃)₂, —CH═CH₂, —CH₂—CH═CH₂,—C≡CH and/or —C≡C—CH₃.

In case the general structure (I) represents monomers or building blocksfor synthesizing the antisense-oligonucleotides of the presentinvention, the terminal groups Y and IL′ are selected independently ofeach other from hydrogen, azido, halogen, cyano, nitro, hydroxy, PG-O—,AG-O—, mercapto, PG-S—, AG-S—, C₁₋₆-alkylthio, amino, PG-N(R^(H))—,AG-N(R^(H))—, mono- or di(C₁₋₆-alkyl)amino, optionally substitutedC₁₋₆-alkoxy, optionally substituted C₁₋₆-alkyl, optionally substitutedC₂₋₆-alkenyl, optionally substituted C₂₋₆-alkenyloxy, optionallysubstituted C₂₋₆-alkynyl, optionally substituted C₂₋₆-alkynyloxy,monophosphate, monothiophosphate, diphosphate, dithiophosphatetriphosphate, trithiophosphate, carboxy, sulphono, hydroxymethyl,PG-O—CH₂—, AG-O—CH₂—, aminomethyl, PG-N(R^(H))—CH₂₋, AG-N(R^(H))—CH₂₋,carboxymethyl, sulphonomethyl, where PG is a protection group for —OH,—SH, and —NH(R^(H)), respectively, AG is an activation group for —OH,—SH, and —NH(R^(H)), respectively, and R^(H) is selected from hydrogenand C₁₋₆-alkyl.

The protection groups PG of hydroxy substituents comprise substitutedtrityl, such as 4,4′-dimethoxytrityl (DMT), 4-monomethoxytrityl (MMT),optionally substituted 9-(9-phenyl)xanthenyl (pixyl), optionallysubstituted methoxytetrahydropyranyl (mthp), silyl such astrimethylsilyl (TMS), triisopropylsilyl (TIPS), tert-butyldimethylsilyl(TBDMS), triethylsilyl, and phenyldimethylsilyl, tert-butylethers,acetals (including two hydroxy groups), acyl such as acetyl or halogensubstituted acetyls, e.g. chloroacetyl or fluoroacetyl, isobutyryl,pivaloyl, benzoyl and substituted benzoyls, methoxymethyl (MOM), benzylethers or substituted benzyl ethers such as 2,6-dichlorobenzyl(2,6-Cl₂Bzl). Alternatively when Y or IL′ is hydroxyl they may beprotected by attachment to a solid support optionally through a linker.

When Y or IL′ is an amino group, illustrative examples of the aminoprotection groups are fluorenylmethoxycarbonyl (Fmoc),tert-butyloxycarbonyl (BOC), trifluoroacetyl, allyloxycarbonyl (alloc orAOC), benzyloxycarbonyl (Z or Cbz), substituted benzyloxycarbonyls suchas 2-chloro benzyloxycarbonyl (2-ClZ), monomethoxytrityl (MMT),dimethoxytrityl (DMT), phthaloyl, and 9-(9-phenyl)xanthenyl (pixyl).

Act represents an activation group for —OH, —SH, and —NH(R^(H)),respectively. Such activation groups are, for instance, selected fromoptionally substituted O-phosphoramidite, optionally substitutedO-phosphortriester, optionally substituted O-phosphordiester, optionallysubstituted H-phosphonate, and optionally substituted O-phosphonate.

In the present context, the term “phosphoramidite” means a group of theformula —P(OR^(x))—N(R^(y))₂, wherein R^(x) designates an optionallysubstituted alkyl group, e.g. methyl, 2-cyanoethyl, or benzyl, and eachof R^(y) designate optionally substituted alkyl groups, e.g. ethyl orisopropyl, or the group —N(R^(y))₂ forms a morpholino group(—N(CH₂CH₂)₂O). R^(x) preferably designates 2-cyanoethyl and the twoR^(y) are preferably identical and designate isopropyl. Thus, anespecially relevant phosphoramidite isN,N-diisopropyl-O-(2-cyanoethyl)-phosphoramidite.

LNA Monomers or LNA Building Blocks

The LNA monomers or LNA building blocks used as starting materials inthe synthesis of the antisense-oligonucleotides of the present inventionare preferably LNA nucleosides of the following general formulae:

The LNA building blocks are normally provided as LNA phosphoramiditeswith the four different nucleobases: adenine (A), guanine (G),5-methyl-cytosine (C*) and thymine (T). The antisense-oligonucleotidesof the present invention containing LNA units are synthesized bystandard phosphoramidite chemistry. In the LNA building blocks thenucleobases are protected. A preferred protecting group for the aminogroup of the purin base is a benzoyl group (Bz), indicated as A^(Bz). Apreferred protecting group for the amino group of the5-methylpyrimidinone base is a benzoyl group (Bz), indicated as C*^(Bz).A preferred protecting group for the amino group of the purinone base isa dimethylformamidine (DMF) group, a diethylformamidine (DEF), adipropylformamidine (DPF), a dibutylformamidine (DBF), or a iso-butyryl(—CO—CH(CH₃)₂) group, indicated as G^(DMF), G^(DEF), G^(DPF), G^(DBF),or G^(iBu). Thus the group -NDMF refers to —N═CH—N(CH₃)₂. DMT refers to4,4′-dimethoxytrityl.

Thus, LNA-T refers to5′-O-(4,4′-dimethoxytrityl)-3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite-thymidineLNA. LNA-C*^(Bz) refers to5′-O-(4,4′-dimethoxytrityl)-3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite-4-N-benzoyl-5-methyl-2′-cytidineLNA. LNA-A^(Bz) refers to5′-O-(4,4′-dimethoxytrityl)-3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite-6-N-benzoyl-2′-adenosineLNA. LNA-G^(DMF) refers to5′-O-(4,4′-dimethoxytrityl)-3′-O-(2-cyanoethyl-N,N-diisopropyl)-phosphoramidite-2-N-dimethylformamidine-2′-guanosineLNA. LNA-G^(iBu) refers to5′-O-(4,4′-dimethoxytrityl)-3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite-2-N-butyryl-2′-guanosineLNA.

Terminal Groups

In case Y represents the 5′-terminal group of anantisense-oligonucleotide of the present invention, the residue Y isalso named Y^(5′) and represents: —OH, —O—C₁₋₆-alkyl, —S—C₁₋₆-alkyl,—O—C₆₋₉-phenyl, —O—C₇₋₁₀-benzyl, —NH—C₁₋₆-alkyl, —N(C₁₋₆-alkyl)₂,—O—C₂₋₆-alkenyl, —S—C₂₋₆-alkenyl, —NH—C₂₋₆-alkenyl, —N(C₂₋₆-alkenyl)₂,—O—C₂₋₆-alkynyl, —S—C₂₋₆-alkynyl, —NH—C₂₋₆-alkynyl, —N(C₂₋₆-alkynyl)₂,—O—C₁₋₆-alkylenyl-O—C₁₋₆-alkyl, —O—[C₁₋₆-alkylenyl-O]m-C₁₋₆-alkyl,—O—CO—C₁₋₆-alkyl, —O—CO—C₂₋₆-alkenyl, —O—CO—C₂₋₆-alkynyl,—O—S(O)—C₁₋₆-alkyl, —O—SO₂—C₁₋₆-alkyl, —O—SO₂—O—C₁₋₆-alkyl,—O—P(O)(O⁻)₂, —O—P(O)(O⁻)(O—C₁₋₆-alkyl), —O—P(O)(O—C₁₋₆-alkyl)₂,—O—P(O)(S⁻)₂, —O—P(O)(S—C₁₋₆-alkyl)₂, —O—P(O)(S⁻)(O—C₁₋₆-alkyl),—O—P(O)(O⁻)(N H—C₁₋₆-alkyl), —O—P(O)(O—C₁₋₆-alkyl)(NH—C₁₋₆-alkyl), —O—P(O)(O⁻)[N(C₁₋₆-alkyl)₂], —O—P(O)(O—C₁₋₆-alkyl)[N(C₁₋₆-alkyl)₂],—O—P(O)(O⁻)(BH₃ ⁻), —O—P(O)(O—C₁₋₆-alkyl)(BH₃ ⁻),—O—P(O)(O⁻)(O—C₁₋₆-alkylenyl-O—C₁₋₆-alkyl),—O—P(O)(O—C₁₋₆-alkylenyl-O—C₁₋₆-alkyl)₂,—O—P(O)(O⁻)(O—C₁₋₆-alkylenyl-S—C₁₋₆-alkyl),—O—P(O)(O—C₁₋₆-alkylenyl-S—C₁₋₆-alkyl)₂,—O—P(O)(O⁻)(OCH₂CH₂O—C₁₋₆-alkyl), —O—P(O)(OCH₂CH₂O—C₁₋₆-alkyl)₂,—O—P(O)(O⁻)(OCH₂CH₂S—C₁₋₆-alkyl), —O—P(O)(OCH₂CH₂S—C₁₋₆-alkyl)₂,—O—P(O)(O⁻)OC₃H₆OH, —O—P(O)(S⁻)OC₃H₆OH, —O—P(S)(S⁻)OC₃H₆OH,

wherein the C₁₋₆-alkyl, C₂₋₆-alkenyl, C₂₋₆-alkynyl, —O—C₆₋₉-phenyl or—O—C₇₋₁₀-benzyl may be further substituted by —F, —OH, C₁₋₄-alkyl,C₂₋₄-alkenyl and/or C₂₋₄-alkynyl where m is selected from 1, 2, 3, 4, 5,6, 7, 8, 9 or 10.

More preferred are: —OCH₃, —OC₂H₅, —OC₃H₇, —O-cyclo-C₃H₅, —OCH(CH₃)₂,—OC(CH₃)₃, —OC₄H₉, —OPh, —OCH₂-Ph, —O—COCH₃, —O—COC₂H₅, —O—COC₃H₇,—O—CO-cyclo-C₃H₅, —O—COCH(CH₃)₂, —OCF₃, —O—S(O)CH₃, —O—S(O)C₂H₅,—O—S(O)C₃H₇, —O—S(O)-cyclo-C₃H₅, —O—SO₂CH₃, —O—SO₂C₂H₅, —O—SO₂C₃H₇,—O—SO₂-cyclo-C₃H₅, —O—SO₂—OCH₃, —O—SO₂—OC₂H₅, —O—SO₂—OC₃H₇,—O—SO₂—O-cyclo-C₃H₅, —O(CH₂)_(n)N[(CH₂)_(n)OH],—O(CH₂)_(n)N[(CH₂)_(n)—H], —O—P(O)(O⁻)OC₃H₆OH, —O—P(O)(S⁻)OC₃H₆OH,

even more preferred are:—OCH₃, —OC₂H₅, —OCH₂CH₂OCH₃ (also known as MOE), —OCH₂CH₂—N(CH₃)₂ (alsoknown as DMAOE), —O[(CH₂)_(n)O]_(m)CH₃, —O(CH₂)_(n)OCH₃, —O(CH₂)_(n)NH₂,—O(CH₂)_(n)N(CH₃)₂, —O—P(O)(O⁻)OC₃H₆OH, —O—P(O)(S⁻)OC₃H₆OH,where n is selected from 1, 2, 3, 4, 5, or 6; andwhere m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In case IL′ represents the 3′-terminal group of anantisense-oligonucleotide of the present invention, the residue IL′ isalso named IL′^(3′) and represents:

—OH, —O—C₁₋₆-alkyl, —S—C₁₋₆-alkyl, —O—C₆₋₉-phenyl, —O—C₇₋₁₀-benzyl,—NH—C₁₋₆-alkyl, —N(C₁₋₆-alkyl)₂, —O—C₂₋₆-alkenyl, —S—C₂₋₆-alkenyl,—NH—C₂₋₆-alkenyl, —N(C₂₋₆-alkenyl)₂, —O—C₂₋₆-alkynyl, —S—C₂₋₆-alkynyl,—NH—C₂₋₆-alkynyl, —N(C₂₋₆-alkynyl)₂, —O—C₁₋₆-alkylenyl-O—C₁₋₆-alkyl,—O—[C₁₋₆-alkylenyl-O]m-C₁₋₆-alkyl, —O—CO—C₁₋₆-alkyl, —O—CO—C₂₋₆-alkenyl,—O—CO—C₂₋₆-alkynyl, —O—S(O)—C₁₋₆-alkyl, —O—SO₂—C₁₋₆-alkyl,—O—SO₂—O—C₁₋₆-alkyl,—O—P(O)(O⁻)₂, —O—P(O)(O⁻)(O—C₁₋₆-alkyl), —O—P(O)(O—C₁₋₆-alkyl)₂,—O—P(O)(S⁻)₂, —O—P(O)(S—C₁₋₆-alkyl)₂, —O—P(O)(S⁻)(O—C₁₋₆-alkyl),—O—P(O)(O⁻)(NH—C₁₋₆-alkyl), —O—P(O)(O—C₁₋₆-alkyl)(NH—C₁₋₆-alkyl),—O—P(O)(O⁻)[N(C₁₋₆-alkyl)₂], —O—P(O)(O—C₁₋₆-alkyl)[N(C₁₋₆-alkyl)₂],—O—P(O)(O⁻)(BH₃ ⁻), —O—P(O)(O—C₁₋₆-alkyl)(BH₃ ⁻),—O—P(O)(O⁻)(O—C₁₋₆-alkylenyl-O—C₁₋₆-alkyl),—O—P(O)(O—C₁₋₆-alkylenyl-O—C₁₋₆-alkyl)₂,—O—P(O)(O⁻)(O—C₁₋₆-alkylenyl-S—C₁₋₆-alkyl),—O—P(O)(O—C₁₋₆-alkylenyl-S—C₁₋₆-alkyl)₂,—O—P(O)(O⁻)(OCH₂CH₂O—C₁₋₆-alkyl), —O—P(O)(OCH₂CH₂O—C₁₋₆-alkyl)₂,—O—P(O)(O⁻)(OCH₂CH₂S—C₁₋₆-alkyl), —O—P(O)(OCH₂CH₂S—C₁₋₆-alkyl)₂,—O—P(O)(O⁻)OC₃H₆OH, —O—P(O)(S⁻)OC₃H₆OH,wherein the C₁₋₆-alkyl, C₂₋₆-alkenyl, C₂₋₆-alkynyl, —O—C₆₋₉-phenyl or—O—C₇₋₁₀-benzyl may be further substituted by —F, —OH, C₁₋₄-alkyl,C₂₋₄-alkenyl and/or C₂₋₄-alkynyl where m is selected from 1, 2, 3, 4, 5,6, 7, 8, 9 or 10.

More preferred are: —OCH₃, —OC₂H₅, —OC₃H₇, —O-cyclo-C₃H₅, —OCH(CH₃)₂,—OC(CH₃)₃, —OC₄H₉, —OPh, —OCH₂-Ph, —O—COCH₃, —O—COC₂H₅, —O—COC₃H₇,—O—CO-cyclo-C₃H₅, —O—COCH(CH₃)₂, —OCF₃, —O—S(O)CH₃, —O—S(O)C₂H₅,—O—S(O)C₃H₇, —O—S(O)-cyclo-C₃H₅, —O—SO₂CH₃, —O—SO₂C₂H₅, —O—SO₂C₃H₇,—O—SO₂-cyclo-C₃H₅, —O—SO₂—OCH₃, —O—SO₂—OC₂H₅, —O—SO₂—OC₃H₇,—O—SO₂—O-cyclo-C₃H₅, —O(CH₂)_(n)N[(CH₂)_(n)OH],—O(CH₂)_(n)N[(CH₂)_(n)—H], —O—P(O)(O⁻)OC₃H₆OH, —O—P(O)(S⁻)OC₃H₆OH,

even more preferred are:—OCH₃, —OC₂H₅, —OCH₂CH₂OCH₃ (also known as MOE), —OCH₂CH₂—N(CH₃)₂ (alsoknown as DMAOE), —O[(CH₂)_(n)O]_(m)CH₃, —O(CH₂)_(n)OCH₃, —O(CH₂)_(n)NH₂,—O(CH₂)_(n)N(CH₃)₂, —O—P(O)(O⁻)OC₃H₆OH, —O—P(O)(S⁻)OC₃H₆OH,where n is selected from 1, 2, 3, 4, 5, or 6; andwhere m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

Preferred LNAs

In preferred embodiments LNA units used in theantisense-oligonucleotides of the present invention preferably have thestructure of general formula (II):

The moiety —C(R^(a)R^(b))—X— represents preferably —C(R^(a)R^(b))—O—,—C(R^(a)R^(b))—NR^(c)—, —C(R^(a)R^(b))—S—, and—C(R^(a)R^(b))—C(R^(a)R^(b))—O—, wherein the substituents R^(a), R^(b)and R^(c) have the meanings as defined herein and are preferablyC₁₋₆-alkyl and more preferably C₁₋₄-alkyl. More preferably—C(R^(a)R^(b))—X— is selected from —CH₂—O—, —CH₂—S—, —CH₂—NH—,—CH₂—N(CH₃)—, —CH₂—CH₂—O—, or —CH₂—CH₂—S—, and more preferably —CH₂—O—,—CH₂—S—, —CH₂—CH₂—O—, or —CH₂—CH₂—S—, and still more preferably —CH₂—O—,—CH₂—S—, or —CH₂—CH₂—O—, and still more preferably —CH₂—O— or —CH₂—S—,and most preferably —CH₂—O—.

All chiral centers and asymmetric substituents (if any) can be either inR or in S orientation. For example, two exemplary stereochemical isomersare the beta-D and alpha-L isoforms as shown below:

Preferred LNA units are selected from general formula (b¹) to (b⁹):

The term “thio-LNA” comprises a locked nucleotide in which X in thegeneral formula (II) is selected from —S— or —CH₂—S—. Thio-LNA can be inboth beta-D and alpha-L-configuration.

The term “amino-LNA” comprises a locked nucleotide in which X in thegeneral formula (II) is selected from —NH—, —N(R)—, —CH₂—NH—, and—CH₂—N(R)—, where R is selected from hydrogen and C₁₋₄-alkyl. Amino-LNAcan be in both beta-D and alpha-L-configuration.

The term “oxy-LNA” comprises a locked nucleotide in which X in thegeneral formula (II) is —O—. Oxy-LNA can be in both beta-D andalpha-L-configuration.

The term “ENA” comprises a locked nucleotide in which X in the generalformula (II) is —CH₂—O— (where the oxygen atom of —CH₂—O— is attached tothe 2′-position relative to the base B). R^(a) and R^(b) areindependently of each other hydrogen or methyl.

In preferred exemplary embodiments LNA is selected from beta-D-oxy-LNA,alpha-L-oxy-LNA, beta-D-amino-LNA and beta-D-thio-LNA, in particularbeta-D-oxy-LNA.

Still more preferred are the following antisense-oligonucleotides (Table1):

SP L Seq ID No. Sequence, 5′-3′ 89 17 102a GCGAGTGACTCACTCAA 90 15 103aCGAGTGACTCACTCA 90 16 104a GCGAGTGACTCACTCA 90 17 105a CGCGAGTGACTCACTCA91 14 106a CGAGTGACTCACTC 91 16 107a CGCGAGTGACTCACTC 91 17 108aGCGCGAGTGACTCACTC 92 14 109a GCGAGTGACTCACT 92 16 110a GCGCGAGTGACTCACT92 17 111a CGCGCGAGTGACTCACT 93 12 112a CGAGTGACTCAC 93 13 113aGCGAGTGACTCAC 93 14 114a CGCGAGTGACTCAC 93 16 115a CGCGCGAGTGACTCAC 9317 116a GCGCGCGAGTGACTCAC 94 13 117a CGCGAGTGACTCA 94 14 118aGCGCGAGTGACTCA 94 15 119a CGCGCGAGTGACTCA 94 16 120a GCGCGCGAGTGACTCA 9417 121a TGCGCGCGAGTGACTCA 95 14 122a CGCGCGAGTGACTC 95 16 123aTGCGCGCGAGTGACTC 95 17 124a GTGCGCGCGAGTGACTC 96 13 125a CGCGCGAGTGACT97 14 126a TGCGCGCGAGTGAC 97 16 127a CGTGCGCGCGAGTGAC 98 13 128aTGCGCGCGAGTGA 107 16 129a GTCGTCGCTCCGTGCG 108 15 130a GTCGTCGCTCCGTGC108 17 131a GTGTCGTCGCTCCGTGC 109 13 132a TCGTCGCTCCGTG 109 15 133aTGTCGTCGCTCCGTG 110 12 134a TCGTCGCTCCGT 110 13 135a GTCGTCGCTCCGT 11014 136a TGTCGTCGCTCCGT 110 15 137a GTGTCGTCGCTCCGT 110 16 138aGGTGTCGTCGCTCCGT 351 16 139a CGTCATAGACCGAGCC 351 12 140a ATAGACCGAGCC354 16 141a GCTCGTCATAGACCGA 354 13 142a CGTCATAGACCGA 355 14 143aCTCGTCATAGACCG 355 15 144a GCTCGTCATAGACCG 356 14 145a GCTCGTCATAGACC381 17 146a CAGCCCCCGACCCATGG 382 16 147a CAGCCCCCGACCCATG 383 14 148aAGCCCCCGACCCAT 384 14 149a CAGCCCCCGACCCA 422 17 150a CGCGTCCACAGGACGAT425 14 151a CGCGTCCACAGGAC 429 15 152a CGATACGCGTCCACA 431 13 153aCGATACGCGTCCA 431 16 154a TGGCGATACGCGTCCA 432 12 155a CGATACGCGTCC 43213 156a GCGATACGCGTCC 432 17 157a GCTGGCGATACGCGTCC 433 15 158aCTGGCGATACGCGTC 433 12 159a GCGATACGCGTC 433 16 160a GCTGGCGATACGCGTC433 14 161a TGGCGATACGCGTC 434 13 162a TGGCGATACGCGT 434 14 163aCTGGCGATACGCGT 434 12 164a GGCGATACGCGT 435 13 165a CTGGCGATACGCG 435 12166a TGGCGATACGCG 437 17 167a ATCGTGCTGGCGATACG 449 16 168aCGTGCGGTGGGATCGT 449 17 169a ACGTGCGGTGGGATCGT 450 17 170aAACGTGCGGTGGGATCG 452 15 171a AACGTGCGGTGGGAT 452 17 172aTGAACGTGCGGTGGGAT 459 17 173a CGACTTCTGAACGTGCG 941 17 174aTTAACGCGGTAGCAGTA 941 16 175a TAACGCGGTAGCAGTA 942 17 176aGTTAACGCGGTAGCAGT 943 15 177a TTAACGCGGTAGCAG 944 13 178a TAACGCGGTAGCA945 12 179a TAACGCGGTAGC 945 13 180a TTAACGCGGTAGC 946 12 181aTTAACGCGGTAG 946 13 182a GTTAACGCGGTAG 946 15 183a CGGTTAACGCGGTAG 94616 184a CCGGTTAACGCGGTAG 947 14 185a CGGTTAACGCGGTA 947 13 186aGGTTAACGCGGTA 947 15 187a CCGGTTAACGCGGTA 947 16 188a GCCGGTTAACGCGGTA947 17 189a TGCCGGTTAACGCGGTA 948 13 190a CGGTTAACGCGGT 949 13 191aCCGGTTAACGCGG 949 14 192a GCCGGTTAACGCGG 949 15 193a TGCCGGTTAACGCGG 95013 194a GCCGGTTAACGCG 950 15 195a CTGCCGGTTAACGCG 950 16 196aGCTGCCGGTTAACGCG 1387 16 197a ATGCCGCGTCAGGTAC 1392 13 198aACATGCCGCGTCA 1393 16 199a GATGACATGCCGCGTC 1394 12 200a GACATGCCGCGT1394 15 201a GATGACATGCCGCGT 1395 13 202a ATGACATGCCGCG 1805 17 203aTCCCGCACCTTGGAACC 1851 16 204a CGATCTCTCAACACGT 1851 17 205aTCGATCTCTCAACACGT 1852 15 206a CGATCTCTCAACACG 1852 16 207aTCGATCTCTCAACACG 1852 17 208a CTCGATCTCTCAACACG 2064 16 209aGTAGTGTTTAGGGAGC 2072 16 210a GCTATTTGGTAGTGTT 2284 15 211aAGCTTATCCTATGAC 2285 14 212a AGCTTATCCTATGA 2355 17 213aCAGGCATTAATAAAGTG 4120 16 214a CTAGGCGCCTCTATGC 4121 14 215aTAGGCGCCTCTATG 4121 15 216a CTAGGCGCCTCTATG 4122 13 217a TAGGCGCCTCTAT4217 16 218a CATGAATGGACCAGTA SP: start position or start nucleotide onSeq. ID No. 2 L: length of the sequence

The antisense-oligonucleotides as disclosed herein such as theantisense-oligonucleotides of Tables 1 to 3 and especially theantisense-oligonucleotides of Tables 4 to 9 consist of nucleotides,preferably DNA nucleotides, which are non-LNA units (also named hereinnon-LNA nucleotides) as well as LNA units (also named herein LNAnucleotides).

Although not explicitly indicated, the antisense-oligonucleotides of thesequences Seq. ID No.s 102a-218a of Table 1 comprise 2 to 4 LNAnucleotides (LNA units) at the 3′ terminus and 2 to 4 LNA nucleotides(LNA units) at the 5′ terminus. Although not explicitly indicated, the“C” in Table 2 which refer to LNA units preferably contain5-methylcytosine (C*) as nucleobase.

That means, as long as not explicitly indicated, theantisense-oligonucleotides of the present invention or as disclosedherein by the letter code A, C, G, T and U may contain anyinternucleotide linkage, any end group and any nucleobase as disclosedherein. Moreover the antisense-oligonucleotides of the present inventionor as disclosed herein are gapmers of any gapmer structure as disclosedherein with at least one LNA unit at the 3′ terminus and at least oneLNA unit at the 5′ terminus. Moreover any LNA unit as disclosed hereincan be used within the antisense-oligonucleotides of the presentinvention or as disclosed herein. Thus, for instance, theantisense-oligonucleotide GCTCGTCATAGACCGA (Seq. ID No. 13) orCGATACGCGTCCACAG (Seq. ID No. 14) or GTAGTGTTTAGGGAGC (Seq. ID No. 15)or GCTATTTGGTAGTGTT (Seq. ID No. 16) or CATGAATGGACCAGTA (Seq. ID No.17) or AGGCATTAATAAAGTG (Seq. ID No. 18) contains at least one LNA unitat the 5′ terminus and at least one LNA unit at the 3′ terminus, anynucleobase, any 3′ end group, any 5′ end group, any gapmer structure,and any internucleotide linkage as disclosed herein and covers alsosalts and optical isomers of that antisense-oligonucleotide.

The use of LNA units is preferred especially at the 3′ terminal and the5′ terminal end. Thus it is preferred if the last 1-5 nucleotides at the3′ terminal end and also the last 1-5 nucleotides at the 5′ terminal endespecially of the sequences disclosed herein and particularly of Seq. IDNo.s 102a-218a of Table 1 are LNA units (also named LNA nucleotides)while in between the 1-5 LNA units at the 3′ and 5′ end 2-14, preferably3-12, more preferably 4-10, more preferably 5-9, still more preferably6-8, non-LNA units (also named non-LNA nucleotides) are present. Suchkind of antisense-oligonucleotides are called gapmers and are disclosedin more detail below. More preferred are 2-5 LNA nucleotides at the 3′end and 2-5 LNA nucleotides at the 5′ end or 1-4 LNA nucleotides at the3′ end and 1-4 LNA nucleotides at the 5′ end and still more preferredare 2-4 LNA nucleotides at the 3′ end and 2-4 LNA nucleotides at the 5′end of the antisense-oligonucleotides with a number of preferably 4-10,more preferably 5-9, still more preferably 6-8 non-LNA units in betweenthe LNA units at the 3′ and the 5′ end.

Moreover as internucleotide linkages between the LNA units and betweenthe LNA units and the non-LNA units, the use of phosphorothioates orphosphorodithioates and preferably phosphorothioates is preferred.

Thus further preferred are antisense-oligonucleotides wherein more than50%, preferably more than 60%, more preferably more than 70%, still morepreferably more than 80%, and most preferably more than 90% of theinternucleotide linkages are phosphorothioates or phosphates and morepreferably phosphorothioate linkages and wherein the last 1-4 or 2-5nucleotides at the 3′ end are LNA units and the last 1-4 or 2-5nucleotides at the 5′ end are LNA units and between the LNA units at theends a sequence of 6-14 nucleotides, preferably 7-12, preferably 8-11,more preferably 8-10 are present which are non-LNA units, preferably DNAunits. Moreover it is preferred that these antisense-oligonucleotides inform of gapmers consist in total of 12 to 20, preferably 12 to 18nucleotides.

Gapmers

The antisense-oligonucleotides of the invention may consist ofnucleotide sequences which comprise both DNA nucleotides which arenon-LNA units as well as LNA nucleotides, and may be arranged in theform of a gapmer.

Thus, the antisense-oligonucleotides of the present invention arepreferably gapmers. A gapmer consists of a middle part of DNA nucleotideunits which are not locked, thus which are non-LNA units. The DNAnucleotides of this middle part could be linked to each other by theinternucleotide linkages (IL) as disclosed herein which preferably maybe phosphate groups, phosphorothioate groups or phosphorodithioategroups and which may contain nucleobase analogues such as 5-propynylcytosine, 7-methylguanine, 7-methyladenine, 2-aminoadenine,2-thiothymine, 2-thiocytosine, or 5-methylcytosine. That DNA units orDNA nucleotides are not bicyclic pentose structures. The middle part ofnon-LNA units is flanked at the 3′ end and the 5′ end by sequencesconsisting of LNA units. Thus gapmers have the general formula:

LNA sequence 1-non-LNA sequence-LNA sequence 2

or

region A-region B-region C

The middle part of the antisense-oligonucleotide which consists of DNAnucleotide units which are non-LNA units is, when formed in a duplexwith the complementary target RNA, capable of recruiting RNase. The 3′and 5′ terminal nucleotide units are LNA units which are preferably inalpha-L configuration, particularly preferred being beta-D-oxy-LNA andalpha-L-oxy LNAs.

Thus, a gapmer is an antisense-oligonucleotide which comprises acontiguous stretch of DNA nucleotides which is capable of recruiting anRNase, such as RNaseH, such as a region of at least 6 or 7 DNAnucleotides which are non-LNA units, referred to herein as middle partor region B, wherein region B is flanked both 5′ and 3′ by regions ofaffinity enhancing nucleotide analogues which are LNA units, such asbetween 1-6 LNA units 5′ and 3′ to the contiguous stretch of DNAnucleotides which is capable of recruiting RNase—these flanking regionsare referred to as regions A and C respectively.

Preferably the gapmer comprises a (poly)nucleotide sequence of formula(5′ to 3′), A- B-C, or optionally A-B-C-D or D-A-B-C, wherein; region A(5′ region) consists of at least one nucleotide analogue, such as atleast one LNA unit, such as between 1-6 LNA units, and region B consistsof at least five consecutive DNA nucleotides which are non-LNA units andwhich are capable of recruiting RNase (when formed in a duplex with acomplementary RNA molecule, such as the mRNA target), and region C(3′region) consists of at least one nucleotide analogue, such as atleast one LNA unit, such as between 1-6 LNA units, and region D, whenpresent consists of 1, 2 or 3 DNA nucleotide units which are non-LNAunits.

In some embodiments, region A consists of 1, 2, 3, 4, 5 or 6 LNA units,such as between 2-5 LNA units, such as 3 or 4 LNA units; and/or region Cconsists of 1, 2, 3, 4, 5 or 6 LNA units, such as between 2-5 LNA units,such as 3 or 4 LNA units. In some embodiments B consists of 5, 6, 7, 8,9, 10, 11 or 12 consecutive DNA nucleotides which are capable ofrecruiting RNase, or between 6-10, or between 7-9, such as 8 consecutivenucleotides which are capable of recruiting RNase. In some embodimentsregion B consists of at least one DNA nucleotide unit, such as 1-12 DNAnucleotide units, preferably between 4-12 DNA nucleotide units, morepreferably between 6-10 DNA nucleotide units, still more preferred suchas between 7-10 DNA nucleotide units, and most preferably 8, 9 or 10 DNAnucleotide units which are non-LNA units.

In some embodiments region A consist of 3 or 4 LNA, region B consists of7, 8, 9 or 10 DNA nucleotide units, and region C consists of 3 or 4 LNAunits. Such designs include (A-B-C): 1-7-2, 2-7-1, 2-7-2, 3-7-1, 3-7-2,1-7-3, 2-7-3, 3-7-3, 2-7-4, 3-7-4, 4-7-2, 4-7-3, 4-7-4, 1-8-1, 1-8-2,2-8-1, 2-8-2, 1-8-3, 3-8-1, 3-8-3, 2-8-3, 3-8-2, 4-8-1, 4-8-2, 1-8-4,2-8-4, 3-8-4, 4-8-3, 4-8-4, 1-9-1, 1-9-2, 2-9-1, 2-9-2, 2-9-3, 3-9-2,3-9-3, 1-9-3, 3-9-1, 4-9-1, 1-9-4, 4-9-2, 2-9-4, 4-9-3, 3-9-4, 4-9-4,1-10-1, 1-10-2, 2-10-1, 2-10-2, 1-10-3, 3-10-1, 2-10-2, 2-10-3, 3-10-2,3-10-3, 2-10-4, 4-10-2, 3-10-4, 4-10-3, 4-10-4, 1-11-1, 1-11-2, 2-11-1,2-11-2, 1-11-3, 3-11-1, 2-11-2, 2-11-3, 3-11-2, 3-11-3, 2-11-4, 4-11-2,3-11-4, 4-11-3, 4-11-4, and may further include region D, which may haveone or 2 DNA nucleotide units, which are non-LNA units.

Further gapmer designs are disclosed in WO2004/046160A and are herebyincorporated by reference. U.S. provisional application, 60/977,409,hereby incorporated by reference, refers to ‘shortmer’ gapmerantisense-oligonucleotide, which are also suitable for the presentinvention.

In some embodiments the antisense-oligonucleotide consists of acontiguous nucleotide sequence of a total of 10, 11, 12, 13 or 14nucleotide units (LNA units and non-LNA units together), wherein thecontiguous nucleotide sequence is of formula (5′-3′), A-B-C, oroptionally A-B-C-D or D-A-B-C, wherein A consists of 1, 2 or 3 LNAunits, and B consists of 7, 8 or 9 contiguous DNA nucleotide units whichare non-LNA units and which are capable of recruiting RNase when formedin a duplex with a complementary RNA molecule (such as a mRNA target),and C consists of 1, 2 or 3 LNA units. When present, D consists of asingle DNA nucleotide unit which is a non-LNA unit.

In some embodiments A consists of 1 LNA unit. In some embodiments Aconsists of 2 LNA units. In some embodiments A consists of 3 LNA units.In some embodiments C consists of 1 LNA unit. In some embodiments Cconsists of 2 LNA units. In some embodiments C consists of 3 LNA units.In some embodiments B consists of 7 DNA nucleotide units which arenon-LNA units. In some embodiments B consists of 8 DNA nucleotide units.In some embodiments B consists of 9 DNA nucleotide units. In someembodiments B consists of 1-9 DNA nucleotide units, such as 2, 3, 4, 5,6, 7 or 8 DNA nucleotide units. The DNA nucleotide units are alwaysnon-LNA units. In some embodiments B comprises 1, 2 or 3 LNA units whichare preferably in the alpha-L configuration and which are morepreferably alpha-L-oxy LNA units. In some embodiments the number ofnucleotides present in A-B-C are selected from the group consisting of(LNA units-region B-LNA units and more preferably alpha-L-oxy LNA units(region A)-region B-(region C) alpha-L-oxy LNA units): 1-8-1, 1-8-2,2-8-1, 2-8-2, 1-8-3, 3-8-1, 3-8-3, 2-8-3, 3-8-2, 4-8-1, 4-8-2, 1-8-4,2-8-4, 3-8-4, 4-8-3, 4-8-4, 1-9-1, 1-9-2, 2-9-1, 2-9-2, 2-9-3, 3-9-2,3-9-3, 1-9-3, 3-9-1, 4-9-1, 1-9-4, 4-9-2, 2-9-4, 4-9-3, 3-9-4, 4-9-4,1-10-1, 1-10-2, 2-10-1, 2-10-2, 1-10-3, 3-10-1, 2-10-2, 2-10-3, 3-10-2,3-10-3, 2-10-4, 4-10-2, 3-10-4, 4-10-3, 4-10-4, 1-11-1, 1-11-2, 2-11-1,2-11-2, 1-11-3, 3-11-1, 2-11-2, 2-11-3, 3-11-2, 3-11-3, 2-11-4, 4-11-2,3-11-4, 4-11-3, 4-11-4. In further preferred embodiments the number ofnucleotides in A-B-C are selected from the group consisting of: 3-8-3,4-8-2, 2-8-4, 3-8-4, 4-8-3, 4-8-4, 3-9-3, 4-9-2, 2-9-4, 4-9-3, 3-9-4,4-9-4, 3-10-3, 2-10-4, 4-10-2, 3-10-4, 4-10-3, 4-10-4, 2-11-4, 4-11-2,3-11-4, 4-11-3 and still more preferred are: 3-8-3, 3-8-4, 4-8-3, 4-8-4,3-9-3, 4-9-3, 3-9-4, 4-9-4, 3-10-3, 3-10-4, 4-10-3, 4-10-4, 3-11-4, and4-11-3.

Phosphorothioate, phosphate or phosphorodithioate and especiallyphosphorothioate internucleotide linkages are also preferred,particularly for the gapmer region B. Phosphorothioate, phosphate orphosphorodithioate linkages and especially phosphorothioateinternucleotide linkages may also be used for the flanking regions (Aand C, and for linking A or C to D, and within region D, if present).

Regions A, B and C, may however comprise internucleotide linkages otherthan phosphorothioate or phosphorodithioate, such as phosphodiesterlinkages, particularly, for instance when the use of nucleotideanalogues protects the internucleotide linkages within regions A and Cfrom endo-nuclease degradation—such as when regions A and C consist ofLNA units.

The internucleotide linkages in the antisense-oligonucleotide may bephosphodiester, phosphorothioate, phosphorodithioate or boranophosphateso as to allow RNase H cleavage of targeted RNA. Phosphorothioate orphosphorodithioate is preferred, for improved nuclease resistance andother reasons, such as ease of manufacture. In one aspect of theoligomer of the invention, the LNA units and/or the non-LNA units arelinked together by means of phosphorothioate groups.

It is recognized that the inclusion of phosphodiester linkages, such asone or two linkages, into an otherwise phosphorothioateantisense-oligonucleotide, particularly between or adjacent to LNA units(typically in region A and or C) can modify the bioavailability and/orbio-distribution of an antisense-oligonucleotide (see WO2008/053314Awhich is hereby incorporated by reference).

In some embodiments, such as in the sequences of theantisense-oligonucleotides disclosed herein and where suitable and notspecifically indicated, all remaining internucleotide linkage groups areeither phosphodiester groups or phosphorothioate groups, or a mixturethereof.

In some embodiments all the internucleotide linkage groups arephosphorothioate groups. When referring to specific gapmerantisense-oligonucleotide sequences, such as those provided herein, itwill be understood that, in various embodiments, when the linkages arephosphorothioate linkages, alternative linkages, such as those disclosedherein may be used, for example phosphate (also named phosphodiester)linkages may be used, particularly for linkages between nucleotideanalogues, such as LNA units. Likewise, when referring to specificgapmer antisense-oligonucleotide sequences, such as those providedherein, when the C residues are annotated as 5′-methyl modifiedcytosine, in various embodiments, one or more of the Cs present in theoligomer may be unmodified C residues.

Legend

As used herein the abbreviations b, d, s, ss have the following meaning:

-   b LNA unit or LNA nucleotide (any one selected from b¹-b⁷)-   b¹ β-D-oxy-LNA-   b² β-D-thio-LNA-   b³ β-D-amino-LNA-   b⁴ α-L-oxy-LNA-   b⁵ β-D-ENA-   b⁶ β-D-(NH)-LNA-   b⁷ β-D-(NCH₃)-LNA-   d 2-deoxy, that means 2-deoxyribose units (e.g. formula B3 or B5    with R═—H)-   C* methyl-C(5-methylcytosine); [consequently dC* is    5-methyl-2′-deoxycytidine]-   A* 2-aminoadenine [consequently dA* is 2-amino-2′-deoxyadenosine]-   s the internucleotide linkage is a phosphorothioate group    (—O—P(O)(S⁻)—O—)-   ss the internucleotide linkage is a phosphorodithioate group    (—O—P(S)(S⁻)—O—)-   /5SpC3/—O—P(O)(O⁻)OC₃H₆OH at 5′-terminal group of an    antisense-oligonucleotide-   /3SpC3/—O—P(O)(O⁻)OC₃H₆OH at 3′-terminal group of an    antisense-oligonucleotide-   /5SpC3s/—O—P(O)(S⁻)OC₃H₆OH at 5′-terminal group of an    antisense-oligonucleotide-   /3SpC3s/—O—P(O)(S⁻)OC₃H₆OH at 3′-terminal group of an    antisense-oligonucleotide    nucleotides in bold are LNA nucleotides    nucleotides not in bold are non-LNA nucleotides

Gapmer Sequences

The following antisense-oligonucleotides in form of gapmers as listed inTable 2 to Table 9 and more preferably in Table 4 to 9 are especiallypreferred.

TABLE 2 SP L Seq ID No. Sequence, 5′-3′ 89 17 102bGbsCbsGbsAbsdGsdTsdGsdAsdCsdTsdCsdAsdCsTbsCbsAbsAb 90 15 103bCbsGbsAbsdGsdTsdGsdAsdCsdTsdCsdAsdCsTbsCbsAb 90 16 104bGbsCbsGbsdAsdGsdTsdGsdAsdCsdTsdCsdAsdCsTbsCbsAb 90 17 105bCbsGbsCbsGbsdAsdGsdTsdGsdAsdCsdTsdCsdAsCbsTbsCbsAb 91 14 106bCbsGbsAbsdGsdTsdGsdAsdCsdTsdCsdAsCbsTbsCb 91 16 107bCbsGbsCbsdGsdAsdGsdTsdGsdAsdCsdTsdCsdAsCbsTbsCb 91 17 108bGbsCbsGbsCbsdGsdAsdGsdTsdGsdAsdCsdTsdCsAbsCbsTbsCb 92 14 109bGbsCbsGbsdAsdGsdTsdGsdAsdCsdTsdCsAbsCbsTb 92 16 110bGbsCbsGbsdCsdGsdAsdGsdTsdGsdAsdCsdTsdCsAbsCbsTb 92 17 111bCbsGbsCbsGbsdCsdGsdAsdGsdTsdGsdAsdCsdTsCbsAbsCbsTb 93 12 112bCbsGbsdAsdGsdTsdGsdAsdCsdTsdCsAbsCb 93 13 113bGbsCbsGbsdAsdGsdTsdGsdAsdCsdTsdCsAbsCb 93 14 114bCbsGbsCbsdGsdAsdGsdTsdGsdAsdCsdTsCbsAbsCb 93 16 115bCbsGbsCbsdGsdCsdGsdAsdGsdTsdGsdAsdCsdTsCbsAbsCb 93 17 116bGbsCbsGbsCbsdGsdCsdGsdAsdGsdTsdGsdAsdCsTbsCbsAbsCb 94 13 117bCbsGbsCbsdGsdAsdGsdTsdGsdAsdCsdTsCbsAb 94 14 118bGbsCbsGbsdCsdGsdAsdGsdTsdGsdAsdCsTbsCbsAb 94 15 119bCbsGbsCbsdGsdCsdGsdAsdGsdTsdGsdAsdCsTbsCbsAb 94 16 120bGbsCbsGbsdCsdGsdCsdGsdAsdGsdTsdGsdAsdCsTbsCbsAb 94 17 121bTbsGbsCbsGbsdCsdGsdCsdGsdAsdGsdTsdGsdAsCbsTbsCbsAb 95 14 122bCbsGbsCbsdGsdCsdGsdAsdGsdTsdGsdAsCbsTbsCb 95 16 123bTbsGbsCbsdGsdCsdGsdCsdGsdAsdGsdTsdGsdAsCbsTbsCb 95 17 124bGbsTbsGbsCbsdGsdCsdGsdCsdGsdAsdGsdTsdGsAbsCbsTbsCb 96 13 125bCbsGbsCbsdGsdCsdGsdAsdGsdTsdGsdAsCbsTb 97 14 126bTbsGbsCbsdGsdCsdGsdCsdGsdAsdGsdTsGbsAbsCb 97 16 127bCbsGbsTbsdGsdCsdGsdCsdGsdCsdGsdAsdGsdTsGbsAbsCb 98 13 128bTbsGbsCbsdGsdCsdGsdCsdGsdAsdGsdTsGbsAb 107 16 129bGbsTbsCbsdGsdTsdCsdGsdCsdTsdCsdCsdGsdTsGbsCbsGb 108 15 130bGbsTbsCbsdGsdTsdCsdGsdCsdTsdCsdCsdGsTbsGbsCb 108 17 131bGbsTbsGbsTbsdCsdGsdTsdCsdGsdCsdTsdCsdCsGbsTbsGbsCb 109 13 132bTbsCbsGbsdTsdCsdGsdCsdTsdCsdCsdGsTbsGb 109 15 133bTbsGbsTbsdCsdGsdTsdCsdGsdCsdTsdCsdCsGbsTbsGb 110 12 134bTbsCbsdGsdTsdCsdGsdCsdTsdCsdCsGbsTb 110 13 135bGbsTbsCbsdGsdTsdCsdGsdCsdTsdCsdCsGbsTb 110 14 136bTbsGbsTbsdCsdGsdTsdCsdGsdCsdTsdCsCbsGbsTb 110 15 137bGbsTbsGbsdTsdCsdGsdTsdCsdGsdCsdTsdCsCbsGbsTb 110 16 138bGbsGbsTbsdGsdTsdCsdGsdTsdCsdGsdCsdTsdCsCbsGbsTb 351 16 139bCbsGbsTbsdCsdAsdTsdAsdGsdAsdCsdCsdGsdAsGbsCbsCb 351 12 140bAbsTbsdAsdGsdAsdCsdCsdGsdAsdGsCbsCb 354 16 141bGbsCbsTbsdCsdGsdTsdCsdAsdTsdAsdGsdAsdCsCbsGbsAb 354 13 142bCbsGbsTbsdCsdAsdTsdAsdGsdAsdCsdCsGbsAb 355 14 143bCbsTbsdCsdGsdTsdCsdAsdTsdAsdGsdAsCbsCbsGb 355 14 143cCbsTbsCbsdGsdTsdCsdAsdTsdAsdGsdAsdCsCbsGb 355 14 143dCbsTbsCbsdGsdTsdCsdAsdTsdAsdGsdAsCbsCbsGb 355 15 144bGbsCbsTbsdCsdGsdTsdCsdAsdTsdAsdGsdAsCbsCbsGb 356 14 145bGbsCbsTbsdCsdGsdTsdCsdAsdTsdAsdGsAbsCbsCb 381 17 146bCbsAbsGbsCbsdCsdCsdCsdCsdGsdAsdCsdCsdCsAbsTbsGbsGb 382 16 147bCbsAbsGbsdCsdCsdCsdCsdCsdGsdAsdCsdCsdCsAbsTbsGb 382 16 147cCbsAbsGbsdCsdCsdCsdCsdCsdGsdAsdCsdCsdCsAsTsG 382 16 147dCbsAbsGbsdCsdCsdCsdCsdCsdGsdAsdCsdCsCbsAbsTbsGb 382 16 147eCbsAbsGbsCbsdCsdCsdCsdCsdGsdAsdCsdCsdCsAbsTbsGb 382 16 147fCbsAbsGbsCbsdCsdCsdCsdCsdGsdAsdCsdCsCbsAbsTbsGb 383 14 148bAbsGbsdCsdCsdCsdCsdCsdGsdAsdCsdCsCbsAbsTb 383 14 148cAbsGbsCbsdCsdCsdCsdCsdGsdAsdCsdCsdCsAbsTb 383 14 148dAbsGbsCbsdCsdCsdCsdCsdGsdAsdCsdCsCbsAbsTb 384 14 149CbsAbsGbsdCsdCsdCsdCsdCsdGsdAsdCsCbsCbsAb 422 17 150bCbsGbsCbsGbsdTsdCsdCsdAsdCsdAsdGsdGsdAsCbsGbsAbsTb 425 14 151bCbsGbsCbsdGsdTsdCsdCsdAsdCsdAsdGsGbsAbsCb 429 15 152bCbsGbsAbsdTsdAsdCsdGsdCsdGsdTsdCsdCsAbsCbsAb 429 15 152cCbsGbsAbsdTsdAsdCsdGsdCsdGsdTsdCsCbsAbsCbsAb 429 15 152dCbsGbsAbsTbsdAsdCsdGsdCsdGsdTsdCsdCsAbsCbsAb 432 12 155bCbsGbsdAsdTsdAsdCsdGsdCsdGsdTsCbsCb 431 13 153bCbsGbsAbsdTsdAsdCsdGsdCsdGsdTsdCsCbsAb 431 13 153cCbsGbsdAsdTsdAsdCsdGsdCsdGsdTsCbsCbsAb 431 16 154bTbsGbsGbsdCsdGsdAsdTsdAsdCsdGsdCsdGsdTsCbsCbsAb 432 12 155cCbsGbsdAsdTsdAsdCsdGsdCsdGsdTsdCsCb 432 12 155dCbsdGsdAsdTsdAsdCsdGsdCsdGsdTsCbsCb 432 13 156bGbsCbsGbsdAsdTsdAsdCsdGsdCsdGsdTsCbsCb 432 17 157bGbsCbsTbsGbsdGsdCsdGsdAsdTsdAsdCsdGsdCsGbsTbsCbsCb 433 15 158bCbsTbsGbsdGsdCsdGsdAsdTsdAsdCsdGsdCsGbsTbsCb 433 12 159bGbsCbsdGsdAsdTsdAsdCsdGsdCsdGsTbsCb 433 16 160bGbsCbsTbsdGsdGsdCsdGsdAsdTsdAsdCsdGsdCsGbsTbsCb 433 14 161bTbsGbsGbsdCsdGsdAsdTsdAsdCsdGsdCsGbsTbsCb 434 12 164bGbsGbsdCsdGsdAsdTsdAsdCsdGsdCsGbsTb 434 13 162bTbsGbsGbsdCsdGsdAsdTsdAsdCsdGsdCsGbsTb 434 13 162cTbsGbsdGsdCsdGsdAsdTsdAsdCsdGsCbsGbsTb 434 14 163bCbsTbsGbsdGsdCsdGsdAsdTsdAsdCsdGsCbsGbsTb 435 13 165bCbsTbsGbsdGsdCsdGsdAsdTsdAsdCsdGsCbsGb 435 12 166bTbsGbsdGsdCsdGsdAsdTsdAsdCsdGsCbsGb 437 17 167bAbsTbsCbsGbsdTsdGsdCsdTsdGsdGsdCsdGsdAsTbsAbsCbsGb 449 16 168bCbsGbsTbsdGsdCsdGsdGsdTsdGsdGsdGsdAsdTsCbsGbsTb 449 17 169bAbsCbsGbsTbsdGsdCsdGsdGsdTsdGsdGsdGsdAsTbsCbsGbsTb 450 17 170bAbsAbsCbsGbsdTsdGsdCsdGsdGsdTsdGsdGsdGsAbsTbsCbsGb 452 15 171bAbsAbsCbsdGsdTsdGsdCsdGsdGsdTsdGsdGsGbsAbsTb 452 17 172bTbsGbsAbsAbsdCsdGsdTsdGsdCsdGsdGsdTsdGsGbsGbsAbsTb 459 17 173bCbsGbsAbsCbsdTsdTsdCsdTsdGsdAsdAsdCsdGsTbsGbsCbsGb 941 17 174bTbsTbsAbsAbsdCsdGsdCsdGsdGsdTsdAsdGsdCsAbsGbsTbsAb 941 16 175bTbsAbsAbsdCsdGsdCsdGsdGsdTsdAsdGsdCsdAsGbsTbsAb 942 17 176bGbsTbsTbsAbsdAsdCsdGsdCsdGsdGsdTsdAsdGsCbsAbsGbsTb 943 15 177bTbsTbsAbsdAsdCsdGsdCsdGsdGsdTsdAsdGsCbsAbsGb 944 13 178bTbsAbsAbsdCsdGsdCsdGsdGsdTsdAsdGsCbsAb 945 12 179bTbsAbsdAsdCsdGsdCsdGsdGsdTsdAsGbsCb 945 13 180bTbsTbsAbsdAsdCsdGsdCsdGsdGsdTsdAsGbsCb 946 12 181bTbsTbsdAsdAsdCsdGsdCsdGsdGsdTsAbsGb 946 13 182bGbsTbsTbsdAsdAsdCsdGsdCsdGsdGsdTsAbsGb 946 15 183bCbsGbsGbsdTsdTsdAsdAsdCsdGsdCsdGsdGsTbsAbsGb 946 16 184bCbsCbsGbsdGsdTsdTsdAsdAsdCsdGsdCsdGsdGsTbsAbsGb 947 14 185bCbsGbsGbsdTsdTsdAsdAsdCsdGsdCsdGsGbsTbsAb 947 13 186bGbsGbsTbsdTsdAsdAsdCsdGsdCsdGsdGsTbsAb 947 15 187bCbsCbsGbsdGsdTsdTsdAsdAsdCsdGsdCsdGsGbsTbsAb 947 16 188bGbsCbsCbsdGsdGsdTsdTsdAsdAsdCsdGsdCsdGsGbsTbsAb 947 17 189bTbsGbsCbsCbsdGsdGsdTsdTsdAsdAsdCsdGsdCsGbsGbsTbsAb 948 13 190bCbsGbsGbsdTsdTsdAsdAsdCsdGsdCsdGsGbsTb 949 13 191bCbsCbsGbsdGsdTsdTsdAsdAsdCsdGsdCsGbsGb 949 14 192bGbsCbsCbsdGsdGsdTsdTsdAsdAsdCsdGsCbsGbsGb 949 15 193bTbsGbsCbsdCsdGsdGsdTsdTsdAsdAsdCsdGsCbsGbsGb 950 13 194bGbsCbsCbsdGsdGsdTsdTsdAsdAsdCsdGsCbsGb 950 15 195bCbsTbsGbsdCsdCsdGsdGsdTsdTsdAsdAsdCsGbsCbsGb 950 16 196bGbsCbsTbsdGsdCsdCsdGsdGsdTsdTsdAsdAsdCsGbsCbsGb 1387 16 197bAbsTbsGbsdCsdCsdGsdCsdGsdTsdCsdAsdGsdGsTbsAbsCb 1392 13 198bAbsCbsAbsdTsdGsdCsdCsdGsdCsdGsdTsCbsAb 1393 16 199bGbsAbsTbsdGsdAsdCsdAsdTsdGsdCsdCsdGsdCsGbsTbsCb 1393 16 199cGbsAbsTbsdGsdAsdCsdAsdTsdGsdCsdCsdGsCbsGbsTbsCb 1393 16 199dGbsAbsTbsGbsdAsdCsdAsdTsdGsdCsdCsdGsdCsGbsTbsCb 1393 16 199eGbsAbsTbsGbsdAsdCsdAsdTsdGsdCsdCsdGsCbsGbsTbsCb 1394 12 200bGbsAbsdCsdAsdTsdGsdCsdCsdGsdCsGbsTb 1394 15 201bGbsAbsTbsdGsdAsdCsdAsdTsdGsdCsdCsdGsCbsGbsTb 1395 13 202bAbsTbsGbsdAsdCsdAsdTsdGsdCsdCsdGsCbsGb 1805 17 203bTbsCbsCbsCbsdGsdCsdAsdCsdCsdTsdTsdGsdGsAbsAbsCbsCb 1851 16 204bCbsGbsAbsdTsdCsdTsdCsdTsdCsdAsdAsdCsdAsCbsGbsTb 1851 17 205bTbsCbsGbsAbsdTsdCsdTsdCsdTsdCsdAsdAsdCsAbsCbsGbsTb 1852 15 206bCbsGbsAbsdTsdCsdTsdCsdTsdCsdAsdAsdCsAbsCbsGb 1852 16 207bTbsCbsGbsdAsdTsdCsdTsdCsdTsdCsdAsdAsdCsAbsCbsGb 1852 17 208bCbsTbsCbsGbsdAsdTsdCsdTsdCsdTsdCsdAsdAsCbsAbsCbsGb 2064 16 209bGbsTbsdAsdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsAbsGbsCb 2064 16 209cGbsTbsAbsGbsdTsdGsdTsdTsdTsdAsdGsdGsdGsAbsGbsCb 2064 16 209dGbsTbsAbsdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsAbsGbsCb 2064 16 209eGbsTbsAbsdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsdAsGbsCb 2064 16 209fGbsTbsAbsdGsdTsdGsdTsdTsdTsdAsdGsdGsGbsAbsGbsCb 2064 16 209gGbsTbsAbsGbsdTsdGsdTsdTsdTsdAsdGsdGsGbsAbsGbsCb 2064 16 209hGbsTbsdAsdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsAbsGbsCb 2064 16 209iGbsTbsAbsGbsTbsdGsdTsdTsdTsdAsdGsdGsGbsAbsGbsCb 2064 16 209jGbsTbsAbsGbsdTsdGsdTsdTsdTsdAsdGsGbsGbsAbsGbsCb 2064 16 209kGbsTbsAbsGbsTbsdGsdTsdTsdTsdAsdGsGbsGbsAbsGbsCb 2072 16 210bGbsCbsdTsdAsdTsdTsdTsdGsdGsdTsdAsdGsdTsGbsTbsTb 2072 16 210cGbsCbsTbsdAsdTsdTsdTsdGsdGsdTsdAsdGsdTsdGsTbsTb 2072 16 210dGbsCbsTbsdAsdTsdTsdTsdGsdGsdTsdAsdGsdTsGbsTbsTb 2072 16 210eGbsCbsTbsAbsdTsdTsdTsdGsdGsdTsdAsdGsdTsGbsTbsTb 2072 16 210fGbsCbsTbsdAsdTsdTsdTsdGsdGsdTsdAsdGsTbsGbsTbsTb 2072 16 210gGbsCbsTbsAbsdTsdTsdTsdGsdGsdTsdAsdGsTbsGbsTbsTb 2284 15 211bAbsGbsCbsdTsdTsdAsdTsdCsdCsdTsdAsdTsGbsAbsCb 2284 15 211cAbsGbsCbsdTsdTsdAsdTsdCsdCsdTsdAsTbsGbsAbsCb 2284 15 211dAbsGbsCbsTbsdTsdAsdTsdCsdCsdTsdAsdTsGbsAbsCb 2285 14 212bAbsGbsdCsdTsdTsdAsdTsdCsdCsdTsdAsTbsGbsAb 2285 14 212cAbsGbsCbsdTsdTsdAsdTsdCsdCsdTsdAsdTsGbsAb 2285 14 212dAbsGbsCbsdTsdTsdAsdTsdCsdCsdTsdAsTbsGbsAb 2355 17 213bCbsAbsGbsdGsdCsdAsdTsdTsdAsdAsdTsdAsdAsdAsGbsTbsGb 2355 17 213cCbsAbsGbsGbsdCsdAsdTsdTsdAsdAsdTsdAsdAsdAsGbsTbsGb 2355 17 213dCbsAbsGbsdGsdCsdAsdTsdTsdAsdAsdTsdAsdAsAbsGbsTbsGb 2355 17 213eCbsAbsGbsGbsdCsdAsdTsdTsdAsdAsdTsdAsdAsAbsGbsTbsGb 4217 16 218dCbsAbsTbsdGsdAsdAsdTsdGsdGsdAsdCsdCsdAsGbsTbsAb 4217 16 218eCbsAbsTbsdGsdAsdAsdTsdGsdGsdAsdCsdCsAbsGbsTbsAb 4217 16 218fCbsAbsTbsGbsdAsdAsdTsdGsdGsdAsdCsdCsdAsGbsTbsAb 4217 16 218gCbsAbsTbsGbsdAsdAsdTsdGsdGsdAsdCsdCsAbsGbsTbsAb 4120 16 214CbsTbsAbsdGsdGsdCsdGsdCsdCsdTsdCsdTsdAsTbsGbsCb 4121 14 215bTbsAbsGbsdGsdCsdGsdCsdCsdTsdCsdTsAbsTbsGb 4121 15 216bCbsTbsAbsdGsdGsdCsdGsdCsdCsdTsdCsdTsAbsTbsGb 4122 13 217bTbsAbsGbsdGsdCsdGsdCsdCsdTsdCsdTsAbsTb

TABLE 3 SP L Seq ID No. Sequence, 5′-3′ 2064 16 209mGbsTbsAbsGbsdTsdGsdTsdTsdTsdAsdGsdGsdGsAbsGbsC*b 2064 16 209nGbsTbsAbsdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsAbsGbsC*b 2064 16 209oGbsTbsAbsdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsdAsGbsC*b 2064 16 209pGbsTbsAbsdGsdTsdGsdTsdTsdTsdAsdGsdGsGbsAbsGbsC*b 2064 16 209qGbsTbsAbsGbsdTsdGsdTsdTsdTsdAsdGsdGsGbsAbsGbsC*b 2064 16 209rGbsTbsdAsdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsAbsGbsC*b 429 15 152eC*bsGbsAbsTbsdAsdC*sdGsdC*sdGsdTsdC*sdC*sAbsC*bsAb 4217 16 −218j C*bsAbsTbsdGsdAsdAsdTsdGsdGsdAsdC*sdC*sAbsGbsTbsAb 2355 17 213fC*bsAbsGbsdGsdC*sdAsdTsdTsdAsdAsdTsdAsdAsAbsGbsTbsGb 2355 17 213gC*bsAbsGbsdGsdC*sdAsdTsdTsdAsdAsdTsdAsdAsdAsGbsTbsGb 432 12 155eC*bsGbsdAsdTsdAsdC*sdGsdC*sdGsdTsC*bsC*b 4217 16 218hC*bsAbsTbsGbsdAsdAsdTsdGsdGsdAsdC*sdC*sAbsGbsTbsAb 2072 16 210hGbsC*bsTbsAbsdTsdTsdTsdGsdGsdTsdAsdGsdTsGbsTbsTb 2072 16 210iGbsC*bsdTsdAsdTsdTsdTsdGsdGsdTsdAsdGsdTsGbsTbsTb 432 12 155fC*bsGbsdAsdTsdAsdC*sdGsdC*sdGsdTsdC*sC*b 2072 16 210jGbsC*bsTbsdAsdTsdTsdTsdGsdGsdTsdAsdGsdTsdGsTbsTb 432 12 155gC*bsdGsdAsdTsdAsdC*sdGsdC*sdGsdTsC*bsC*b 431 13 153dC*bsGbsAbsdTsdAsdC*sdGsdC*sdGsdTsdC*sC*bsAb 429 15 152fC*bsGbsAbsdTsdAsdC*sdGsdC*sdGsdTsdC*sdC*sAbsC*bsAb 4217 16 218iC*bsAbsTbsGbsdAsdAsdTsdGsdGsdAsdC*sdC*sdAsGbsTbsAb 1393 16 199fGbsAbsTbsdGsdAsdC*sdAsdTsdGsdC*sdC*sdGsdC*sGbsTbsC*b 2285 14 212eAbsGbsC*bsdTsdTsdAsdTsdC*sdC*sdTsdAsdTsGbsAb 355 14 143eC*bsTbsdC*sdGsdTsdC*sdAsdTsdAsdGsdAsC*bsC*bsGb 2072 16 210kGbsC*bsTbsdAsdTsdTsdTsdGsdGsdTsdAsdGsdTsGbsTbsTb 1393 16 199gGbsAbsTbsdGsdAsdC*sdAsdTsdGsdC*sdC*sdGsC*bsGbsTbsC*b 2355 17 213hC*bsAbsGbsGbsdC*sdAsdTsdTsdAsdAsdTsdAsdAsAbsGbsTbsGb 429 15 152gC*bsGbsAbsdTsdAsdC*sdGsdC*sdGsdTsdC*sC*bsAbsC*bsAb 2285 14 212fAbsGbsC*bsdTsdTsdAsdTsdC*sdC*sdTsdAsTbsGbsAb 355 14 143fC*bsTbsC*bsdGsdTsdC*sdAsdTsdAsdGsdAsC*bsC*bsGb 1393 16 199hGbsAbsTbsGbsdAsdC*sdAsdTsdGsdC*sdC*sdGsC*bsGbsTbsC*b 1393 16 199iGbsAbsTbsGbsdAsdC*sdAsdTsdGsdC*sdC*sdGsdC*sGbsTbsC*b 4217 16 218kC*bsAbsTbsdGsdAsdAsdTsdGsdGsdAsdC*sdC*sdAsGbsTbsAb 2285 14 212gAbsGbsdC*sdTsdTsdAsdTsdC*sdC*sdTsdAsTbsGbsAb 434 13 162dTbsGbsGbsdC*sdGsdAsdTsdAsdC*sdGsdC*sGbsTb 383 14 148eAbsGbsC*bsdC*sdC*sdC*sdC*sdGsdAsdC*sdC*sdC*sAbsTb 431 13 153eC*bsGbsdAsdTsdAsdC*sdGsdC*sdGsdTsC*bsC*bsAb 2284 15 211eAbsGbsC*bsdTsdTsdAsdTsdC*sdC*sdTsdAsdTsGbsAbsC*b 355 14 143gC*bsTbsC*bsdGsdTsdC*sdAsdTsdAsdGsdAsdC*sC*bsGb 2284 15 211fAbsGbsC*bsdTsdTsdAsdTsdC*sdC*sdTsdAsTbsGbsAbsC*b 383 14 148fAbsGbsC*bsdC*sdC*sdC*sdC*sdGsdAsdC*sdC*sC*bsAbsTb 383 14 148gAbsGbsdC*sdC*sdC*sdC*sdC*sdGsdAsdC*sdC*sC*bsAbsTb 382 16 147gC*bsAbsGbsdC*sdC*sdC*sdC*sdC*sdGsdAsdC*sdC*sdC*sAbsTbsGb 2072 16 210mGbsC*bsTbsdAsdTsdTsdTsdGsdGsdTsdAsdGsTbsGbsTbsTb 2072 16 210nGbsC*bsTbsAbsdTsdTsdTsdGsdGsdTsdAsdGsTbsGbsTbsTb 434 13 162eTbsGbsdGsdC*sdGsdAsdTsdAsdC*sdGsC*bsGbsTb 2284 15 211gAbsGbsC*bsTbsdTsdAsdTsdC*sdC*sdTsdAsdTsGbsAbsC*b 382 16 147hC*bsAbsGbsdC*sdC*sdC*sdC*sdC*sdGsdAsdC*sdC*sC*bsAbsTbsGb 382 16 147iC*bsAbsGbsC*bsdC*sdC*sdC*sdC*sdGsdAsdC*sdC*sdC*sAbsTbsGb 382 16 147jC*bsAbsGbsdC*sdC*sdC*sdC*sdC*sdGsdAsdC*sdC*sdC*sAbsTbsGb 2355 17 213iC*bsAbsGbsGbsdC*sdAsdTsdTsdAsdAsdTsdAsdAsdAsGbsTbsGb 382 16 147kC*bsAbsGbsC*bsdC*sdC*sdC*sdC*sdGsdAsdC*sdC*sC*bsAbsTbsGb

Preferred Antisense-Oligonucleotides

In the following preferred antisense-oligonucleotides of the presentinvention are disclosed.

Thus, the present invention is preferably directed to anantisense-oligonucleotide in form of a gapmer consisting of 10 to 28nucleotides, preferably 11 to 24 nucleotides, more preferably 12 to 20,and still more preferably 13 to 19 or 14 to 18 nucleotides and 1 to 5 ofthese nucleotides at the 5′ terminal end and 1 to 5 nucleotides at the3′ terminal end of the antisense-oligonucleotide are LNA nucleotides andbetween the LNA nucleotides at the 5′ terminal end and the 3′ terminalend a sequence of at least 6, preferably 7 and more preferably 8 DNAnucleotides is present, and the antisense-oligonucleotide is capable ofhybridizing with a region of the gene encoding the TGF-R_(II) or with aregion of the mRNA encoding the TGF-R_(II), wherein theantisense-oligonucleotide is represented by the following sequence5′-N¹-GTCATAGA-N²-3′ (Seq. ID No. 12) or 5′-N³-ACGCGTCC-N⁴-3′ (Seq. IDNo. 98) or 5′-N¹¹-TGTTTAGG-N¹²-3′ (Seq. ID No. 10) or5′-N⁵-TTTGGTAG-N⁶-3′ (Seq. ID No. 11) or 5′-N⁷-AATGGACC-N⁸-3′ (Seq. IDNo. 100) or 5′-N⁹-ATTAATAA-N¹⁰-3′ (Seq. ID No. 101), wherein

N¹ represents: CATGGCAGACCCCGCTGCTC-, ATGGCAGACCCCGCTGCTC-,TGGCAGACCCCGCTGCTC-, GGCAGACCCCGCTGCTC-, GCAGACCCCGCTGCTC-,CAGACCCCGCTGCTC-, AGACCCCGCTGCTC-, GACCCCGCTGCTC-, ACCCCGCTGCTC-,CCCCGCTGCTC-, CCCGCTGCTC-, CCGCTGCTC-, CGCTGCTC-, GCTGCTC-, CTGCTC-,TGCTC-, GCTC-, CTC-, TC-, or C-;

N²

represents: —C, —CC, -CCG, -CCGA, -CCGAG, -CCGAGC, -CCGAGCC, -CCGAGCCC,-CCGAGCCCC, -CCGAGCCCCC, -CCGAGCCCCCA, -CCGAGCCCCCAG, -CCGAGCCCCCAGC,-CCGAGCCCCCAGCG, -CCGAGCCCCCAGCGC, -CCGAGCCCCCAGCGCA,-CCGAGCCCCCAGCGCAG, -CCGAGCCCCCAGCGCAGC, -CCGAGCCCCCAGCGCAGCG, or-CCGAGCCCCCAGCGCAGCGG;N³ represents: GGTGGGATCGTGCTGGCGAT-, GTGGGATCGTGCTGGCGAT-,TGGGATCGTGCTGGCGAT-, GGGATCGTGCTGGCGAT-, GGATCGTGCTGGCGAT-,GATCGTGCTGGCGAT-, ATCGTGCTGGCGAT-, TCGTGCTGGCGAT-, CGTGCTGGCGAT-,GTGCTGGCGAT-, TGCTGGCGAT-, GCTGGCGAT-, CTGGCGAT-, TGGCGAT-, GGCGAT-,GCGAT-, CGAT-, GAT-, AT-, or T-;

N⁴

represents: -ACAGGACGATGTGCAGCGGC, -ACAGGACGATGTGCAGCGG,-ACAGGACGATGTGCAGCG, -ACAGGACGATGTGCAGC, -ACAGGACGATGTGCAG,-ACAGGACGATGTGCA, -ACAGGACGATGTGC, -ACAGGACGATGTG, -ACAGGACGATGT,-ACAGGACGATG, -ACAGGACGAT, -ACAGGACGA, -ACAGGACG, -ACAGGAC, -ACAGGA,-ACAGG, -ACAG, -ACA, -AC, or -A;N⁵ represents: GCCCAGCCTGCCCCAGAAGAGCTA-, CCCAGCCTGCCCCAGAAGAGCTA-,CCAGCCTGCCCCAGAAGAGCTA-, CAGCCTGCCCCAGAAGAGCTA-, AGCCTGCCCCAGAAGAGCTA-,GCCTGCCCCAGAAGAGCTA-, CCTGCCCCAGAAGAGCTA-, CTGCCCCAGAAGAGCTA-,TGCCCCAGAAGAGCTA-, GCCCCAGAAGAGCTA-, CCCCAGAAGAGCTA-, CCCAGAAGAGCTA-,CCAGAAGAGCTA-, CAGAAGAGCTA-, AGAAGAGCTA-, GAAGAGCTA-, AAGAGCTA-,AGAGCTA-, GAGCTA-, AGCTA-, GCTA-, CTA-, TA-, or A-;N⁶ represents: -TGTTTAGGGAGCCGTCTTCAGGAA, -TGTTTAGGGAGCCGTCTTCAGGA,-TGTTTAGGGAGCCGTCTTCAGG, -TGTTTAGGGAGCCGTCTTCAG, -TGTTTAGGGAGCCGTCTTCA,-TGTTTAGGGAGCCGTCTTC, -TGTTTAGGGAGCCGTCTT, -TGTTTAGGGAGCCGTCT,-TGTTTAGGGAGCCGTC, -TGTTTAGGGAGCCGT, -TGTTTAGGGAGCCG, -TGTTTAGGGAGCC,-TGTTTAGGGAGC, -TGTTTAGGGAG, -TGTTTAGGGA, -TGTTTAGGG, -TGTTTAGG,-TGTTTAG, -TGTTTA, -TGTTT, -TGTT, -TGT, -TG, or -T;N⁷ represents: TGAATCTTGAATATCTCATG-, GAATCTTGAATATCTCATG-,AATCTTGAATATCTCATG-, ATCTTGAATATCTCATG-, TCTTGAATATCTCATG-,CTTGAATATCTCATG-, TTGAATATCTCATG-, TGAATATCTCATG-, GAATATCTCATG-,AATATCTCATG-, ATATCTCATG-, TATCTCATG-, ATCTCATG-, TCTCATG-, CTCATG-,TCATG-, CATG-, ATG-, TG-, or G-;

N⁸

represents: -AGTATTCTAGAAACTCACCA, -AGTATTCTAGAAACTCACC,-AGTATTCTAGAAACTCAC, -AGTATTCTAGAAACTCA, -AGTATTCTAGAAACTC,-AGTATTCTAGAAACT, -AGTATTCTAGAAAC, -AGTATTCTAGAAA, -AGTATTCTAGAA,-AGTATTCTAGA, -AGTATTCTAG, -AGTATTCTA, -AGTATTCT, -AGTATTC, -AGTATT,-AGTAT, -AGTA, -AGT, -AG, or -A;N⁹ represents: ATTCATATTTATATACAGGC-, TTCATATTTATATACAGGC-,TCATATTTATATACAGGC-, CATATTTATATACAGGC-, ATATTTATATACAGGC-,TATTTATATACAGGC-, ATTTATATACAGGC-, TTTATATACAGGC-, TTATATACAGGC-,TATATACAGGC-, ATATACAGGC-, TATACAGGC-, ATACAGGC-, TACAGGC-, ACAGGC-,CAGGC-, AGGC-, GGC-, GC-, or C-;

N¹⁰

represents: -AGTGCAAATGTTATTGGCTA, -AGTGCAAATGTTATTGGCT,-AGTGCAAATGTTATTGGC, -AGTGCAAATGTTATTGG, -AGTGCAAATGTTATTG,-AGTGCAAATGTTATT, -AGTGCAAATGTTAT, -AGTGCAAATGTTA, -AGTGCAAATGTT,-AGTGCAAATGT, -AGTGCAAATG, -AGTGCAAAT, -AGTGCAAA, -AGTGCAA, -AGTGCA,-AGTGC, -AGTG, -AGT, -AG, or -A;N¹¹ represents: TGCCCCAGAAGAGCTATTTGGTAG-, GCCCCAGAAGAGCTATTTGGTAG-,CCCCAGAAGAGCTATTTGGTAG-, CCCAGAAGAGCTATTTGGTAG-, CCAGAAGAGCTATTTGGTAG-,CAGAAGAGCTATTTGGTAG-, AGAAGAGCTATTTGGTAG-, GAAGAGCTATTTGGTAG-,AAGAGCTATTTGGTAG-, AGAGCTATTTGGTAG-, GAGCTATTTGGTAG-, AGCTATTTGGTAG-,GCTATTTGGTAG-, CTATTTGGTAG-, TATTTGGTAG-, ATTTGGTAG-, TTTGGTAG-,TTGGTAG-, TGGTAG-, GGTAG-, GTAG-, TAG-, AG- or G-,N¹² represents: -GAGCCGTCTTCAGGAATCTTCTCC (Seq. ID No. 764),-GAGCCGTCTTCAGGAATCTTCTC, -GAGCCGTCTTCAGGAATCTTCT,-GAGCCGTCTTCAGGAATCTTC, -GAGCCGTCTTCAGGAATCTT, -GAGCCGTCTTCAGGAATCT,-GAGCCGTCTTCAGGAATC, -GAGCCGTCTTCAGGAAT, -GAGCCGTCTTCAGGAA,-GAGCCGTCTTCAGGA, -GAGCCGTCTTCAGG, -GAGCCGTCTTCAG, -GAGCCGTCTTCA,-GAGCCGTCTTC, -GAGCCGTCTT, -GAGCCGTCT, -GAGCCGTC, -GAGCCGT, -GAGCCG,-GAGCC, -GAGC, -GAG, -GA, or -G;or wherein N¹ to N¹² represent any of the limited lists of residues asdisclosed herein, and salts and optical isomers of theantisense-oligonucleotide.

Moreover, the present invention is preferably directed to anantisense-oligonucleotide in form of a gapmer consisting of 10 to 28nucleotides, preferably 11 to 24 nucleotides, more preferably 12 to 20,and still more preferably 13 to 19 or 14 to 18 nucleotides and 1 to 5 ofthese nucleotides at the 5′ terminal end and 1 to 5 nucleotides at the3′ terminal end of the antisense-oligonucleotide are LNA nucleotides andbetween the LNA nucleotides at the 5′ terminal end and the 3′ terminalend a sequence of at least 6, preferably 7 and more preferably 8 DNAnucleotides is present, and the antisense-oligonucleotide is capable ofhybridizing with a region of the gene encoding the TGF-R_(II) or with aregion of the mRNA encoding the TGF-R_(II), wherein theantisense-oligonucleotide is represented by the following sequence5′-N¹-GTCATAGA-N²-3′ (Seq. ID No. 12), wherein N¹ represents:GGCAGACCCCGCTGCTC-, GCAGACCCCGCTGCTC-, CAGACCCCGCTGCTC-,AGACCCCGCTGCTC-, GACCCCGCTGCTC-, ACCCCGCTGCTC-, CCCCGCTGCTC-,CCCGCTGCTC-, CCGCTGCTC-, CGCTGCTC-, GCTGCTC-, CTGCTC-, TGCTC-, GCTC-,CTC-, TC-, or C-;

N²

represents: —C, —CC, -CCG, -CCGA, -CCGAG, -CCGAGC, -CCGAGCC, -CCGAGCC C,-CCGAGCCCC, -CCGAGCCCCC, -CCGAGCCCCCA, -CCGAGCCCCCAG, -CC GAGCCCCCAGC,-CCGAGCCCCCAGCG, -CCGAGCCCCCAGCGC, -CCGAGCCC CCAGCGCA, or-CCGAGCCCCCAGCGCAG;and salts and optical isomers of the antisense-oligonucleotide.

N¹ and/or N² may also represent any of the further limited lists of 3′and 5′ residues as disclosed herein.

Especially preferred gapmer antisense-oligonucleotides falling undergeneral formula S1:

S1 (Seq. ID No. 12) 5′-N¹-GTCATAGA-N²-3′are the following:

(Seq. ID No. 19) CCGCTGCTCGTCATAGAC (Seq. ID No. 20) CGCTGCTCGTCATAGACC(Seq. ID No. 21) GCTGCTCGTCATAGACCG (Seq. ID No. 22) CTGCTCGTCATAGACCGA(Seq. ID No. 23) TGCTCGTCATAGACCGAG (Seq. ID No. 24) GCTCGTCATAGACCGAGC(Seq. ID No. 25) CTCGTCATAGACCGAGCC (Seq. ID No. 26) TCGTCATAGACCGAGCCC(Seq. ID No. 27) CGTCATAGACCGAGCCCC (Seq. ID No. 28) CGCTGCTCGTCATAGAC(Seq. ID No. 29) GCTGCTCGTCATAGACC (Seq. ID No. 30) CTGCTCGTCATAGACCG(Seq. ID No. 31) TGCTCGTCATAGACCGA (Seq. ID No. 32) GCTCGTCATAGACCGAG(Seq. ID No. 33) CTCGTCATAGACCGAGC (Seq. ID No. 34) TCGTCATAGACCGAGCC(Seq. ID No. 35) CGTCATAGACCGAGCCC (Seq. ID No. 36) GCTGCTCGTCATAGAC(Seq. ID No. 37) CTGCTCGTCATAGACC (Seq. ID No. 38) TGCTCGTCATAGACCG(Seq. ID No. 39) GCTCGTCATAGACCGA (Seq. ID No. 40) CTCGTCATAGACCGAG(Seq. ID No. 41) TCGTCATAGACCGAGC (Seq. ID No. 42) CGTCATAGACCGAGCC(Seq. ID No. 43) CTGCTCGTCATAGAC (Seq. ID No. 44) TGCTCGTCATAGACC(Seq. ID No. 45) GCTCGTCATAGACCG (Seq. ID No. 46) CTCGTCATAGACCGA(Seq. ID No. 47) TCGTCATAGACCGAG (Seq. ID No. 48) CGTCATAGACCGAGC(Seq. ID No. 49) TGCTCGTCATAGAC (Seq. ID No. 50) GCTCGTCATAGACC(Seq. ID No. 51) CTCGTCATAGACCG (Seq. ID No. 52) TCGTCATAGACCGA(Seq. ID No. 53) CGTCATAGACCGAG

The antisense-oligonucleotides of formula S1 in form of gapmers (LNAsegment 1-DNA segment-LNA segment 2) contain an LNA segment at the 5′terminal end consisting of 2 to 5, preferably 2 to 4 LNA units andcontain an LNA segment at the 3′ terminal end consisting of 2 to 5,preferably 2 to 4 LNA units and between the two LNA segments one DNAsegment consisting of 6 to 14, preferably 7 to 12 and more preferably 8to 11 DNA units.

The antisense-oligonucleotides of formula S1 contain the LNA nucleotides(LNA units) as disclosed herein, especially these disclosed in thechapter “Locked Nucleic Acids (LNA®)” and preferably these disclosed inthe chapter “Preferred LNAs”. The LNA units and the DNA units maycomprise standard nucleobases such as adenine (A), cytosine (C), guanine(G), thymine (T) and uracil (U), but may also contain modifiednucleobases as disclosed in the chapter “Nucleobases”. Theantisense-oligonucleotides of formula S1 or the LNA segments and the DNAsegment of the antisense-oligonucleotide may contain any internucleotidelinkage as disclosed herein and especially these disclosed in thechapter “Internucleotide Linkages (IL)”. The antisense-oligonucleotidesof formula S1 may optionally also contain endgroups at the 3′ terminalend and/or the 5′ terminal end and especially these disclosed in thechapter “Terminal groups”.

Experiments have shown that modified nucleobases do not considerablyincrease or change the activity of the inventiveantisense-oligonucleotides in regard to tested neurological andoncological indications. The modified nucleobases 5-methylcytosine or2-aminoadenine have been demonstrated to further increase the activityof the antisense-oligonucleotides of formula S1 especially if5-methylcytosine is used in the LNA nucleotides only or in the LNAnucleotides and in the DNA nucleotides and/or if 2-aminoadenine is usedin the DNA nucleotides and not in the LNA nucleotides.

The preferred gapmer structure of the antisense-oligonucleotides offormula S1 is as follows: 3-8-3, 4-8-2, 2-8-4, 3-8-4, 4-8-3, 4-8-4,3-9-3, 4-9-2, 2-9-4, 4-9-3, 3-9-4, 4-9-4, 3-10-3, 2-10-4, 4-10-2,3-10-4, 4-10-3, 4-10-4, 2-11-4, 4-11-2, 3-11-4, 4-11-3 and still morepreferred: 3-8-3, 3-8-4, 4-8-3, 4-8-4, 3-9-3, 4-9-3, 3-9-4, 4-9-4,3-10-3, 3-10-4, 4-10-3, 4-10-4, 3-11-4, and 4-11-3.

As LNA units for the antisense-oligonucleotides of formula S1 especiallyβ-D-oxy-LNA (b¹), β-D-thio-LNA (b²), α-L-oxy-LNA (b⁴), β-D-ENA (b⁵),β-D-(NH)-LNA (b⁶), β-D-(NCH₃)-LNA (b⁷), β-D-(ONH)-LNA (b⁸) andβ-D-(ONCH₃)-LNA (b⁹) are preferred. Experiments have been shown that allof these LNA units b¹, b², b⁴, b⁵, b⁶, b⁷, b⁸, and b⁹ can be synthesizedwith the required effort and lead to antisense-oligonucleotides ofcomparable stability and activity. However based on the experiments theLNA units b¹, b², b⁴, b⁵, b⁶, and b⁷ are further preferred. Stillfurther preferred are the LNA units b¹, b², b⁴, b⁶, and b⁷, and evenmore preferred are the LNA units b¹ and b⁴ and most preferred also inregard to the complexity of the chemical synthesis is the β-D-oxy-LNA(b¹).

So far no special 3′ terminal group or 5′ terminal group could be foundwhich remarkably had changed or increased the stability or activity foroncological or neurological indications, so that 3′ and 5′ end groupsare possible but not explicitly preferred.

Various internucleotide bridges or internucleotide linkages arepossible. In the formulae disclosed herein the internucleotide linkageIL is represented by -IL′-Y—. Thus, IL=—IL′-Y—═—X″—P(═X′)(X⁻)—Y—,wherein IL is preferably selected form the group consisting of:

—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—,—S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—,—O—P(O)(CH₃)—O—, —O—P(O)(OCH₃)—O—, —O—P(O)(NH(CH₃))—O—,—O—P(O)[N(CH₃)₂]—O—, —O—P(O)(BH₃ ⁻)—O—, —O—P(O)(OCH₂CH₂OCH₃)—O—,—O—P(O)(OCH₂CH₂SCH₃)—O—, —O—P(O)(O⁻)—N(CH₃)—, —N(CH₃)—P(O)(O⁻)—O—.

Preferred are the internucleotide linkages IL selected from—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—,—S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—,—O—P(O)(OCH₃)—O—, —O—P(O)(NH(CH₃))—O—, —O—P(O)[N(CH₃)₂]—O—,—O—P(O)(OCH₂CH₂OCH₃)—O—, and more preferred selected from—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—S—,—S—P(O)(S⁻)—O—, —O—P(O)—, —O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—, and still morepreferred selected from —O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—,and most preferably selected from —O—P(O)(O⁻)—O— and —O—P(O)(S⁻)—O—.

Thus, the present invention is preferably directed to anantisense-oligonucleotide in form of a gapmer consisting of 10 to 28nucleotides, preferably 11 to 24 nucleotides, more preferably 12 to 20,and still more preferably 13 to 19 or 14 to 18 nucleotides and 1 to 5 ofthese nucleotides at the 5′ terminal end and 1 to 5 nucleotides at the3′ terminal end of the antisense-oligonucleotide are LNA nucleotides andbetween the LNA nucleotides at the 5′ terminal end and the 3′ terminalend a sequence of at least 6, preferably 7 and more preferably 8 DNAnucleotides is present, and the antisense-oligonucleotide is capable ofhybridizing with a region of the gene encoding the TGF-R_(II) or with aregion of the mRNA encoding the TGF-R_(II), wherein theantisense-oligonucleotide is represented by the following sequence5′-N¹-GTCATAGA-N²-3′ (Seq. ID No. 12), wherein

N¹ represents: GGCAGACCCCGCTGCTC-, GCAGACCCCGCTGCTC-, CAGACCCCGCTGCTC-,AGACCCCGCTGCTC-, GACCCCGCTGCTC-, ACCCCGCTGCTC-, CCCCGCTGCTC-,CCCGCTGCTC-, CCGCTGCTC-, CGCTGCTC-, GCTGCTC-, CTGCTC-, TGCTC-, GCTC-,CTC-, TC-, or C-;

N²

represents: —C, —CC, -CCG, -CCGA, -CCGAG, -CCGAGC, -CCGAGCC, -CCGAGCC C,-CCGAGCCCC, -CCGAGCCCCC, -CCGAGCCCCCA, -CCGAGCCCCCAG, -CC GAGCCCCCAGC,-CCGAGCCCCCAGCG, -CCGAGCCCCCAGCGC, -CCGAGCCC CCAGCGCA, or-CCGAGCCCCCAGCGCAG; andthe LNA nucleotides are selected from β-D-oxy-LNA (b¹), β-D-thio-LNA(b²), α-L-oxy-LNA (b⁴), β-D-ENA (b⁵), β-D-(NH)-LNA (b⁶), β-D-(NCH₃)-LNA(b⁷), β-D-(ONH)-LNA (b⁸) and β-D-(ONCH₃)-LNA (b⁹); and preferably fromβ-D-oxy-LNA (b¹), β-D-thio-LNA (b²), α-L-oxy-LNA (b⁴), β-D-(NH)-LNA(b⁶), and β-D-(NCH₃)-LNA (b⁷); andthe internucleotide linkages are selected from—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—,—S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—,—O—P(O)(CH₃)—O—, —O—P(O)(OCH₃)—O—, —O—P(O)(NH(CH₃))—O—,—O—P(O)[N(CH₃)₂]—O—, —O—P(O)(BH₃ ⁻)—O—, —O—P(O)(OCH₂CH₂OCH₃)—O—,—O—P(O)(OCH₂CH₂SCH₃)—O—, —O—P(O)(O⁻)—N(CH₃)—, —N(CH₃)—P(O)(O⁻)—O—; andpreferably from —O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—,—S—P(O)(O⁻)—S—, —S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—, —S—P(O)(O⁻)—S—;and salts and optical isomers of the antisense-oligonucleotide. Suchpreferred antisense-oligonucleotides may not contain any modified 3′ and5′ terminal end or may not contain any 3′ and 5′ terminal group and mayas modified nucleobase contain 5-methylcytosine and/or 2-aminoadenine.

More preferably N¹ represents: CAGACCCCGCTGCTC-, AGACCCCGCTGCTC-,GACCCCGCTGCTC-, ACCCCGCTGCTC-, CCCCGCTGCTC-, CCCGCTGCTC-, CCGCTGCTC-,CGCTGCTC-, GCTGCTC-, CTGCTC-, TGCTC-, GCTC-, CTC-, TC-, or C-; and

N²

represents: —C, —CC, -CCG, -CCGA, -CCGAG, -CCGAGC, -CCGAGCC, -CCGAGCCC,-CCGAGCCCC, -CCGAGCCCCC, -CCGAGCCCCCA, -CCGAGCCCCCAG, -CC GAGCCCCCAGC,-CCGAGCCCCCAGCG, or -CCGAGCCCCCAGCGC.

Still further preferred, the present invention is directed to anantisense-oligonucleotide in form of a gapmer consisting of 11 to 24nucleotides, more preferably 12 to 20, and still more preferably 13 to19 or 14 to 18 nucleotides and 2 to 5 of these nucleotides at the 5′terminal end and 2 to 5 nucleotides at the 3′ terminal end of theantisense-oligonucleotide are LNA nucleotides and between the LNAnucleotides at the 5′ terminal end and the 3′ terminal end a sequence ofat least 7, preferably at least 8 DNA nucleotides is present, and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the antisense-oligonucleotide is represented by thefollowing sequence 5′-N¹-GTCATAGA-N²-3′ (Seq. ID No. 12), wherein

N¹ represents: ACCCCGCTGCTC-, CCCCGCTGCTC-, CCCGCTGCTC-, CCGCTGCTC-,CGCTGCTC-, GCTGCTC-, CTGCTC-, TGCTC-, GCTC-, CTC-, TC-, or C-;preferably N¹ represents: CCCGCTGCTC-, CCGCTGCTC-, CGCTGCTC-, GCTGCTC-,CTGCTC-, TGCTC-, GCTC-, CTC-, TC-, or C-; and

N²

represents: —C, —CC, -CCG, -CCGA, -CCGAG, -CCGAGC, -CCGAGCC, -CCGAGCC C,-CCGAGCCCC, or -CCGAGCCCCC, -CCGAGCCCCCA, or -CCGAGCCCCCAG; preferablyN² represents: —C, —CC, -CCG, -CCGA, -CCGAG, -CCGAGC, -CCGAGCC, -CCGAGCCC, -CCGAGCCCC, or -CCGAGCCCCC;and the LNA nucleotides are selected from β-D-oxy-LNA (b¹), β-D-thio-LNA(b²), α-L-oxy-LNA (b⁴), β-D-(NH)-LNA (b⁶), and β-D-(NCH₃)-LNA (b⁷); andthe internucleotide linkages are selected from—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—,—S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—; andpreferably selected from phosphate, phosphorothioate andphosphorodithioate;and salts and optical isomers of the antisense-oligonucleotide. Suchpreferred antisense-oligonucleotides may not contain any modified 3′ and5′ terminal end or may not contain any 3′ and 5′ terminal group and mayas modified nucleobase contain 5-methylcytosine and/or 2-aminoadenine.

Especially preferred are the gapmer antisense-oligonucleotides of Seq.ID No. 19 to Seq. ID No. 53 containing a segment of 2 to 5, preferably 2to 4 and more preferably 3 to 4 LNA units at the 3′ terminus and asegment of 2 to 5, preferably 2 to 4 and more preferably 3 to 4 LNAunits at the 5′ terminus and a segment of at least 6, preferably 7 andmore preferably 8 DNA units between the two segments of LNA units,wherein the LNA units are selected from β-D-oxy-LNA (b¹), β-D-thio-LNA(b²), α-L-oxy-LNA (b⁴), β-D-(NH)-LNA (b⁶), and β-D-(NCH₃)-LNA (b⁷) andthe internucleotide linkages are selected from phosphate,phosphorothioate and phosphorodithioate. Such preferredantisense-oligonucleotides may not contain any modified 3′ and 5′terminal end or may not contain any 3′ and 5′ terminal group and may asmodified nucleobase contain 5-methylcytosine in the LNA units,preferably all the LNA units and/or 2-aminoadenine in some or all DNAunits and/or 5-methylcytosine in some or all DNA units.

Also especially preferred are the gapmer antisense-oligonucleotides ofTable 4 (Seq. ID No. 232a to 244b).

Moreover, the present invention is preferably directed to anantisense-oligonucleotide in form of a gapmer consisting of 10 to 28nucleotides, preferably 11 to 24 nucleotides, more preferably 12 to 20,and still more preferably 13 to 19 or 14 to 18 nucleotides and 1 to 5 ofthese nucleotides at the 5′ terminal end and 1 to 5 nucleotides at the3′ terminal end of the antisense-oligonucleotide are LNA nucleotides andbetween the LNA nucleotides at the 5′ terminal end and the 3′ terminalend a sequence of at least 6, preferably 7 and more preferably 8 DNAnucleotides is present, and the antisense-oligonucleotide is capable ofhybridizing with a region of the gene encoding the TGF-R_(II) or with aregion of the mRNA encoding the TGF-R_(II), wherein theantisense-oligonucleotide is represented by the following sequence5′-N³-ACGCGTCC-N⁴-3′ (Seq. ID No. 98), wherein N³ represents:GGGATCGTGCTGGCGAT-, GGATCGTGCTGGCGAT-, GATCGTGCTGGCGAT-,ATCGTGCTGGCGAT-, TCGTGCTGGCGAT-, CGTGCTGGCGAT-, GTGCTGGCGAT-,TGCTGGCGAT-, GCTGGCGAT-, CTGGCGAT-, TGGCGAT-, GGCGAT-, GCGAT-, CGAT-,GAT-, AT-, or T-;

N⁴

represents: -ACAGGACGATGTGCAGC, -ACAGGACGATGTGCAG, -ACAGGACGATGTGCA,-ACAGGACGATGTGC, -ACAGGACGATGTG, -ACAGGACGATGT, -ACAGGACGATG,-ACAGGACGAT, -ACAGGACGA, -ACAGGACG, -ACAGGAC, -ACAGGA, -ACAGG, -ACAG,-ACA, -AC, or -A;and salts and optical isomers of the antisense-oligonucleotide.

N³ and/or N⁴ may also represent any of the further limited lists of 3′and 5′ residues as disclosed herein.

Especially preferred gapmer antisense-oligonucleotides falling undergeneral formula S2:

S2 (Seq. ID No. 98) 5′-N³-ACGCGTCC-N⁴-3′are the following:

(Seq. ID No. 54) GCTGGCGATACGCGTCCA (Seq. ID No. 55) CTGGCGATACGCGTCCAC(Seq. ID No. 56) TGGCGATACGCGTCCACA (Seq. ID No. 57) GGCGATACGCGTCCACAG(Seq. ID No. 58) GCGATACGCGTCCACAGG (Seq. ID No. 59) CGATACGCGTCCACAGGA(Seq. ID No. 60) GATACGCGTCCACAGGAC (Seq. ID No. 61) ATACGCGTCCACAGGACG(Seq. ID No. 62) TACGCGTCCACAGGACGA (Seq. ID No. 63) CTGGCGATACGCGTCCA(Seq. ID No. 64) TGGCGATACGCGTCCAC (Seq. ID No. 65) GGCGATACGCGTCCACA(Seq. ID No. 66) GCGATACGCGTCCACAG (Seq. ID No. 67) CGATACGCGTCCACAGG(Seq. ID No. 68) GATACGCGTCCACAGGA (Seq. ID No. 349) ATACGCGTCCACAGGAC(Seq. ID No. 350) TACGCGTCCACAGGACG (Seq. ID No. 351) TGGCGATACGCGTCCA(Seq. ID No. 352) GGCGATACGCGTCCAC (Seq. ID No. 353) GCGATACGCGTCCACA(Seq. ID No. 354) CGATACGCGTCCACAG (Seq. ID No. 355) GATACGCGTCCACAGG(Seq. ID No. 356) ATACGCGTCCACAGGA (Seq. ID No. 357) TACGCGTCCACAGGAC(Seq. ID No. 358) GGCGATACGCGTCCA (Seq. ID No. 359) GCGATACGCGTCCAC(Seq. ID No. 360) CGATACGCGTCCACA (Seq. ID No. 361) GATACGCGTCCACAG(Seq. ID No. 362) ATACGCGTCCACAGG (Seq. ID No. 363) TACGCGTCCACAGGA(Seq. ID No. 364) GCGATACGCGTCCA (Seq. ID No. 365) CGATACGCGTCCAC(Seq. ID No. 366) GATACGCGTCCACA (Seq. ID No. 367) ATACGCGTCCACAG(Seq. ID No. 368) TACGCGTCCACAGG

The antisense-oligonucleotides of formula S2 in form of gapmers (LNAsegment 1-DNA segment-LNA segment 2) contain an LNA segment at the 5′terminal end consisting of 2 to 5, preferably 2 to 4 LNA units andcontain an LNA segment at the 3′ terminal end consisting of 2 to 5,preferably 2 to 4 LNA units and between the two LNA segments one DNAsegment consisting of 6 to 14, preferably 7 to 12 and more preferably 8to 11 DNA units.

The antisense-oligonucleotides of formula S2 contain the LNA nucleotides(LNA units) as disclosed herein, especially these disclosed in thechapter “Locked Nucleic Acids (LNA®)” and preferably these disclosed inthe chapter “Preferred LNAs”. The LNA units and the DNA units maycomprise standard nucleobases such as adenine (A), cytosine (C), guanine(G), thymine (T) and uracil (U), but may also contain modifiednucleobases as disclosed in the chapter “Nucleobases”. Theantisense-oligonucleotides of formula S2 or the LNA segments and the DNAsegment of the antisense-oligonucleotide may contain any internucleotidelinkage as disclosed herein and especially these disclosed in thechapter “Internucleotide Linkages (IL)”. The antisense-oligonucleotidesof formula S2 may optionally also contain endgroups at the 3′ terminalend and/or the 5′ terminal end and especially these disclosed in thechapter “Terminal groups”.

Experiments have shown that modified nucleobases do not considerablyincrease or change the activity of the inventiveantisense-oligonucleotides in regard to tested neurological andoncological indications. The modified nucleobases 5-methylcytosine or2-aminoadenine have been demonstrated to further increase the activityof the antisense-oligonucleotides of formula S2 especially if5-methylcytosine is used in the LNA nucleotides only or in the LNAnucleotides and in the DNA nucleotides and/or if 2-aminoadenine is usedin the DNA nucleotides and not in the LNA nucleotides.

The preferred gapmer structure of the antisense-oligonucleotides offormula S2 is as follows: 3-8-3, 4-8-2, 2-8-4, 3-8-4, 4-8-3, 4-8-4,3-9-3, 4-9-2, 2-9-4, 4-9-3, 3-9-4, 4-9-4, 3-10-3, 2-10-4, 4-10-2,3-10-4, 4-10-3, 4-10-4, 2-11-4, 4-11-2, 3-11-4, 4-11-3 and still morepreferred: 3-8-3, 3-8-4, 4-8-3, 4-8-4, 3-9-3, 4-9-3, 3-9-4, 4-9-4,3-10-3, 3-10-4, 4-10-3, 4-10-4, 3-11-4, and 4-11-3.

As LNA units for the antisense-oligonucleotides of formula S2 especiallyβ-D-oxy-LNA (b¹), β-D-thio-LNA (b²), α-L-oxy-LNA (b⁴), β-D-ENA (b⁵),β-D-(NH)-LNA (b⁶), β-D-(NCH₃)-LNA (b⁷), β-D-(ONH)-LNA (b⁸) andβ-D-(ONCH₃)-LNA (b⁹) are preferred. Experiments have been shown that allof these LNA units b¹, b², b⁴, b⁵, b⁶, b⁷, b⁸, and b⁹ can be synthesizedwith the required effort and lead to antisense-oligonucleotides ofcomparable stability and activity. However based on the experiments theLNA units b¹, b², b⁴, b⁵, b⁶, and b⁷ are further preferred. Stillfurther preferred are the LNA units b¹, b², b⁴, b⁶, and b⁷, and evenmore preferred are the LNA units b¹ and b⁴ and most preferred also inregard to the complexity of the chemical synthesis is the β-D-oxy-LNA(b¹).

So far no special 3′ terminal group or 5′ terminal group could be foundwhich remarkably had changed or increased the stability or activity foroncological or neurological indications, so that 3′ and 5′ end groupsare possible but not explicitly preferred.

Various internucleotide bridges or internucleotide linkages arepossible. In the formulae disclosed herein the internucleotide linkageIL is represented by -IL′-Y—. Thus, IL=—IL′-Y—═—X″—P(═X′)(X⁻)—Y—,wherein IL is preferably selected form the group consisting of:

—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—,—S—P(O)(S⁻)—(O)—, —O—PO—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—,—O—P(O)(CH₃)—O—, —O—P(O)(OCH₃)—O—, —O—P(O)(NH(CH₃))—O—,—O—P(O)[N(CH₃)₂]—O—, —O—P(O)(BH₃ ⁻)—O—, —O—P(O)(OCH₂CH₂OCH₃)—O—, —O—P(O)(OCH₂CH₂SCH₃)—O—, —O—P(O)(O⁻)—N(CH₃)—, —N(CH₃)—P(O)(O⁻)—O—. Preferredare the internucleotide linkages IL selected from —O—P(O)(O⁻)—O—,—O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—, —S—P(O)(S⁻)—O—,—O—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—, —O—P(O)(OCH₃)—O—,—O—P(O)(NH(CH₃))—O—, —O—P(O)[N(CH₃)₂]—O—, —O—P(O)(OCH₂CH₂OCH₃)—O—, andmore preferred selected from —O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—,—O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—, —S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—,—O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—, and still more preferred selected from—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, and most preferablyselected from —O—P(O)(O⁻)—O— and —O—P(O)(S⁻)—O—.

Thus, the present invention is preferably directed to anantisense-oligonucleotide in form of a gapmer consisting of 10 to 28nucleotides, preferably 11 to 24 nucleotides, more preferably 12 to 20,and still more preferably 13 to 19 or 14 to 18 nucleotides and 1 to 5 ofthese nucleotides at the 5′ terminal end and 1 to 5 nucleotides at the3′ terminal end of the antisense-oligonucleotide are LNA nucleotides andbetween the LNA nucleotides at the 5′ terminal end and the 3′ terminalend a sequence of at least 6, preferably 7 and more preferably 8 DNAnucleotides is present, and the antisense-oligonucleotide is capable ofhybridizing with a region of the gene encoding the TGF-R_(II) or with aregion of the mRNA encoding the TGF-R_(II), wherein theantisense-oligonucleotide is represented by the following sequence5′-N³-ACGCGTCC-N⁴-3′ (Seq. ID No. 98), wherein

N³ represents: GGGATCGTGCTGGCGAT-, GGATCGTGCTGGCGAT-, GATCGTGCTGGCGAT-,ATCGTGCTGGCGAT-, TCGTGCTGGCGAT-, CGTGCTGGCGAT-, GTGCTGGCGAT-,TGCTGGCGAT-, GCTGGCGAT-, CTGGCGAT-, TGGCGAT-, GGCGAT-, GCGAT-, CGAT-,GAT-, AT-, or T-; and

N⁴

represents: -ACAGGACGATGTGCAGC, -ACAGGACGATGTGCAG, -ACAGGACGA TGTGCA,-ACAGGACGATGTGC, -ACAGGACGATGTG, -ACAGGACGATGT, -ACAGGACGATG,-ACAGGACGAT, -ACAGGACGA, -ACAGGACG, -ACAGGAC, -ACAGGA, -ACAGG, -ACAG,-ACA, -AC, or -A, andthe LNA nucleotides are selected from β-D-oxy-LNA (b¹), β-D-thio-LNA(b²), α-L-oxy-LNA (b⁴), β-D-ENA (b⁵), β-D-(NH)-LNA (b⁶), β-D-(NCH₃)-LNA(b⁷), β-D-(ONH)-LNA (b⁸) and β-D-(ONCH₃)-LNA (b⁹); and preferably fromβ-D-oxy-LNA (b¹), β-D-thio-LNA (b²), α-L-oxy-LNA (b⁴), β-D-(NH)-LNA(b⁶), and β-D-(NCH₃)-LNA (b⁷); andthe internucleotide linkages are selected from—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—,—S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—,—O—P(O)(CH₃)—O—, —O—P(O)(OCH₃)—O—, —O—P(O)(NH(CH₃))—O—,—O—P(O)[N(CH₃)₂]—O—, —O—P(O)(BH₃ ⁻)—O—, —O—P(O)(OCH₂CH₂OCH₃)—O—,—O—P(O)(OCH₂CH₂SCH₃)—O—, —O—P(O)(O⁻)—N(CH₃)—, —N(CH₃)—P(O)(O⁻)—O—; andpreferably from —O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—,—S—P(O)(O⁻)—O—, —S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—,—S—P(O)(O⁻)—S—;and salts and optical isomers of the antisense-oligonucleotide. Suchpreferred antisense-oligonucleotides may not contain any modified 3′ and5′ terminal end or may not contain any 3′ and 5′ terminal group and mayas modified nucleobase contain 5-methylcytosine and/or 2-aminoadenine.

More preferably N³ represents: GATCGTGCTGGCGAT-, ATCGTGCTGGCGAT-,TCGTGCTGGCGAT-, CGTGCTGGCGAT-, GTGCTGGCGAT-, TGCTGGCGAT-, GCTGGCGAT-,CTGGCGAT-, TGGCGAT-, GGCGAT-, GCGAT-, CGAT-, GAT-, AT-, or T-; and

N⁴

represents: -ACAGGACGATGTGCA, -ACAGGACGATGTGC, -ACAGGACGATGTG,-ACAGGACGATGT, -ACAGGACGATG, -ACAGGACGAT, -ACAGGACGA, -ACAGGACG,-ACAGGAC, -ACAGGA, -ACAGG, -ACAG, -ACA, -AC, or -A.

Still further preferred, the present invention is directed to anantisense-oligonucleotide in form of a gapmer consisting of 11 to 24nucleotides, more preferably 12 to 20, and still more preferably 13 to19 or 14 to 18 nucleotides and 2 to 5 of these nucleotides at the 5′terminal end and 2 to 5 nucleotides at the 3′ terminal end of theantisense-oligonucleotide are LNA nucleotides and between the LNAnucleotides at the 5′ terminal end and the 3′ terminal end a sequence ofat least 7, preferably at least 8 DNA nucleotides is present, and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the antisense-oligonucleotide is represented by thefollowing sequence 5′-N³-ACGCGTCC-N⁴-3′ (Seq. ID No. 98), wherein

N³ represents: CGTGCTGGCGAT-, GTGCTGGCGAT-, TGCTGGCGAT-, GCTGGCGAT-,CTGGCGAT-, TGGCGAT-, GGCGAT-, GCGAT-, CGAT-, GAT-, AT-, or T-;preferably N³ represents: TGCTGGCGAT-, GCTGGCGAT-, CTGGCGAT-, TGGCGAT-,GGCGAT-, GCGAT-, CGAT-, GAT-, AT-, or T-;and

N⁴

represents: -ACAGGACGATGT, -ACAGGACGATG, -ACAGGACGAT, -ACAGGACG A,-ACAGGACG, -ACAGGAC, -ACAGGA, -ACAGG, -ACAG, -ACA, -AC, or -A;preferably N⁴ represents: -ACAGGACGAT, -ACAGGACGA, -ACAGGACG, -ACAGGAC,-ACAGG A, -ACAGG, -ACAG, -ACA, -AC, or -A; and the LNA nucleotides areselected from β-D-oxy-LNA (b¹), β-D-thio-LNA (b²), α-L-oxy-LNA (b⁴),β-D-(NH)-LNA (b⁶), and β-D-(NCH₃)-LNA (b⁷); and the internucleotidelinkages are selected from—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—,—S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—; andpreferably selected from phosphate, phosphorothioate andphosphorodithioate;and salts and optical isomers of the antisense-oligonucleotide. Suchpreferred antisense-oligonucleotides may not contain any modified 3′ and5′ terminal end or may not contain any 3′ and 5′ terminal group and mayas modified nucleobase contain 5-methylcytosine and/or 2-aminoadenine.

Especially preferred are the gapmer antisense-oligonucleotides of Seq.ID No. 54 to Seq. ID No. 68 and Seq. ID No. 349 to Seq. ID No. 368containing a segment of 2 to 5, preferably 2 to 4 and more preferably 3to 4 LNA units at the 3′ terminus and a segment of 2 to 5, preferably 2to 4 and more preferably 3 to 4 LNA units at the 5′ terminus and asegment of at least 6, preferably 7 and more preferably 8 DNA unitsbetween the two segments of LNA units, wherein the LNA units areselected from β-D-oxy-LNA (b¹), β-D-thio-LNA (b²), α-L-oxy-LNA (b⁴),β-D-(NH)-LNA (b⁶), and β-D-(NCH₃)-LNA (b⁷) and the internucleotidelinkages are selected from phosphate, phosphorothioate andphosphorodithioate. Such preferred antisense-oligonucleotides may notcontain any modified 3′ and 5′ terminal end or may not contain any 3′and 5′ terminal group and may as modified nucleobase contain5-methylcytosine in the LNA units, preferably all the LNA units and/or2-aminoadenine in some or all DNA units and/or 5-methylcytosine in someor all DNA units.

Also especially preferred are the gapmer antisense-oligonucleotides ofTable 5 (Seq. ID No. 245a to 257b).

Moreover, the present invention is preferably directed to anantisense-oligonucleotide in form of a gapmer consisting of 10 to 28nucleotides, preferably 11 to 24 nucleotides, more preferably 12 to 20,and still more preferably 13 to 19 or 14 to 18 nucleotides and 1 to 5 ofthese nucleotides at the 5′ terminal end and 1 to 5 nucleotides at the3′ terminal end of the antisense-oligonucleotide are LNA nucleotides andbetween the LNA nucleotides at the 5′ terminal end and the 3′ terminalend a sequence of at least 6, preferably 7 and more preferably 8 DNAnucleotides is present, and the antisense-oligonucleotide is capable ofhybridizing with a region of the gene encoding the TGF-R_(II) or with aregion of the mRNA encoding the TGF-R_(II), wherein theantisense-oligonucleotide is represented by the following sequence5′-N¹¹-TGTTTAGG-N¹²-3′ (Seq. ID No. 10), wherein

N¹¹ represents: GAAGAGCTATTTGGTAG-, AAGAGCTATTTGGTAG-, AGAGCTATTTGGTAG-,GAGCTATTTGGTAG-, AGCTATTTGGTAG-, GCTATTTGGTAG-, CTATTTGGTAG-,TATTTGGTAG-, ATTTGGTAG-, TTTGGTAG-, TTGGTAG-, TGGTAG-, GGTAG-, GTAG-,TAG-, AG- or G-,N¹² represents: -GAGCCGTCTTCAGGAAT, -GAGCCGTCTTCAGGAA, -GAGCCGTCTTCAGGA,-GAGCCGTCTTCAGG, -GAGCCGTCTTCAG, -GAGCCGTCTTCA, -GAGCCGTCTTC,-GAGCCGTCTT, -GAGCCGTCT, -GAGCCGTC, -GAGCCGT, -GAGCCG, -GAGCC, -GAGC,-GAG, -GA, or -G;and salts and optical isomers of the antisense-oligonucleotide.N¹¹ and/or N¹² may also represent any of the further limited lists of 3′and 5′ residues as disclosed herein.

Especially preferred gapmer antisense-oligonucleotides falling undergeneral formula S3: 5′—N^(1l)-TGTTTAGG-N¹²-3′ (Seq. ID No. 10) S3

S3 (Seq. ID No. 10) 5′-N¹¹-TGTTTAGG-N¹²-3′are the following:

(Seq. ID No. 369) ATTTGGTAGTGTTTAGGG (Seq. ID No. 370)TTTGGTAGTGTTTAGGGA (Seq. ID No. 371) TTGGTAGTGTTTAGGGAG(Seq. ID No. 372) TGGTAGTGTTTAGGGAGC (Seq. ID No. 373)GGTAGTGTTTAGGGAGCC (Seq. ID No. 374) GTAGTGTTTAGGGAGCCG(Seq. ID No. 375) TAGTGTTTAGGGAGCCGT (Seq. ID No. 376)AGTGTTTAGGGAGCCGTC (Seq. ID No. 377) GTGTTTAGGGAGCCGTCT(Seq. ID No. 378) TTTGGTAGTGTTTAGGG (Seq. ID No. 379) TTGGTAGTGTTTAGGGA(Seq. ID No. 380) TGGTAGTGTTTAGGGAG (Seq. ID No. 381) GGTAGTGTTTAGGGAGC(Seq. ID No. 382) GTAGTGTTTAGGGAGCC (Seq. ID No. 383) TAGTGTTTAGGGAGCCG(Seq. ID No. 384) AGTGTTTAGGGAGCCGT (Seq. ID No. 385) GTGTTTAGGGAGCCGTC(Seq. ID No. 386) TTGGTAGTGTTTAGGG (Seq. ID No. 387) TGGTAGTGTTTAGGGA(Seq. ID No. 388) GGTAGTGTTTAGGGAG (Seq. ID No. 389) GTAGTGTTTAGGGAGC(Seq. ID No. 390) TAGTGTTTAGGGAGCC (Seq. ID No. 391) AGTGTTTAGGGAGCCG(Seq. ID No. 392) GTGTTTAGGGAGCCGT (Seq. ID No. 393) TGGTAGTGTTTAGGG(Seq. ID No. 394) GGTAGTGTTTAGGGA (Seq. ID No. 395) GTAGTGTTTAGGGAG(Seq. ID No. 396) TAGTGTTTAGGGAGC (Seq. ID No. 397) AGTGTTTAGGGAGCC(Seq. ID No. 398) GTGTTTAGGGAGCCG (Seq. ID No. 399) GGTAGTGTTTAGGG(Seq. ID No. 400) GTAGTGTTTAGGGA (Seq. ID No. 401) TAGTGTTTAGGGAG(Seq. ID No. 402) AGTGTTTAGGGAGC (Seq. ID No. 403) GTGTTTAGGGAGCC

The antisense-oligonucleotides of formula S3 in form of gapmers (LNAsegment 1-DNA segment-LNA segment 2) contain an LNA segment at the 5′terminal end consisting of 2 to 5, preferably 2 to 4 LNA units andcontain an LNA segment at the 3′ terminal end consisting of 2 to 5,preferably 2 to 4 LNA units and between the two LNA segments one DNAsegment consisting of 6 to 14, preferably 7 to 12 and more preferably 8to 11 DNA units.

The antisense-oligonucleotides of formula S3 contain the LNA nucleotides(LNA units) as disclosed herein, especially these disclosed in thechapter “Locked Nucleic Acids (LNA®)” and preferably these disclosed inthe chapter “Preferred LNAs”. The LNA units and the DNA units maycomprise standard nucleobases such as adenine (A), cytosine (C), guanine(G), thymine (T) and uracil (U), but may also contain modifiednucleobases as disclosed in the chapter “Nucleobases”. Theantisense-oligonucleotides of formula S3 or the LNA segments and the DNAsegment of the antisense-oligonucleotide may contain any internucleotidelinkage as disclosed herein and especially these disclosed in thechapter “Internucleotide Linkages (IL)”. The antisense-oligonucleotidesof formula S3 may optionally also contain endgroups at the 3′ terminalend and/or the 5′ terminal end and especially these disclosed in thechapter “Terminal groups”.

Experiments have shown that modified nucleobases do not considerablyincrease or change the activity of the inventiveantisense-oligonucleotides in regard to tested neurological andoncological indications. The modified nucleobases 5-methylcytosine or2-aminoadenine have been demonstrated to further increase the activityof the antisense-oligonucleotides of formula S3 especially if5-methylcytosine is used in the LNA nucleotides only or in the LNAnucleotides and in the DNA nucleotides and/or if 2-aminoadenine is usedin the DNA nucleotides and not in the LNA nucleotides.

The preferred gapmer structure of the antisense-oligonucleotides offormula S3 is as follows: 3-8-3, 4-8-2, 2-8-4, 3-8-4, 4-8-3, 4-8-4,3-9-3, 4-9-2, 2-9-4, 4-9-3, 3-9-4, 4-9-4, 3-10-3, 2-10-4, 4-10-2,3-10-4, 4-10-3, 4-10-4, 2-11-4, 4-11-2, 3-11-4, 4-11-3 and still morepreferred: 3-8-3, 3-8-4, 4-8-3, 4-8-4, 3-9-3, 4-9-3, 3-9-4, 4-9-4,3-10-3, 3-10-4, 4-10-3, 4-10-4, 3-11-4, and 4-11-3.

As LNA units for the antisense-oligonucleotides of formula S3 especiallyβ-D-oxy-LNA (b¹), β-D-thio-LNA (b²), α-L-oxy-LNA (b⁴), β-D-ENA (b⁵),β-D-(NH)-LNA (b⁶), β-D-(NCH₃)-LNA (b⁷), β-D-(ONH)-LNA (b⁸) andβ-D-(ONCH₃)-LNA (b⁹) are preferred. Experiments have been shown that allof these LNA units b¹, b², b⁴, b⁵, b⁶, b⁷, b⁸, and b⁹ can be synthesizedwith the required effort and lead to antisense-oligonucleotides ofcomparable stability and activity. However based on the experiments theLNA units b¹, b², b⁴, b⁵, b⁶, and b⁷ are further preferred. Stillfurther preferred are the LNA units b¹, b², b⁴, b⁶, and b⁷, and evenmore preferred are the LNA units b¹ and b⁴ and most preferred also inregard to the complexity of the chemical synthesis is the β-D-oxy-LNA(b¹).

So far no special 3′ terminal group or 5′ terminal group could be foundwhich remarkably had changed or increased the stability or activity foroncological or neurological indications, so that 3′ and 5′ end groupsare possible but not explicitly preferred.

Various internucleotide bridges or internucleotide linkages arepossible. In the formulae disclosed herein the internucleotide linkageIL is represented by -IL′-Y—. Thus, IL=—IL′-Y—═—X″—P(═X′)(X⁻)—Y—,wherein IL is preferably selected form the group consisting of:

—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—,—S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—,—O—P(O)(CH₃)—O—, —O—P(O)(OCH₃)—O—, —O—P(O)(NH(CH₃))—O—,—O—P(O)[N(CH₃)₂]—O—, —O—P(O)(BH₃ ⁻)—O—, —O—P(O)(OCH₂CH₂OCH₃)—O—, —O—P(O)(OCH₂CH₂SCH₃)—O—, —O—P(O)(O⁻)—N(CH₃)—, —N(CH₃)—P(O)(O⁻)—O—. Preferredare the internucleotide linkages IL selected from —O—P(O)(O⁻)—O—,—O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—, —S—P(O)(S⁻)—O—,—O—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—, —O—P(O)(OCH₃)—O—,—O—P(O)(NH(CH₃))—O—, —O—P(O)[N(CH₃)₂]—O—, —O—P(O)(OCH₂CH₂OCH₃)—O—, andmore preferred selected from —O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—,—O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—S—, —S—P(O)(S⁻)—O—, —O—P(O)—,—O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—, and still more preferred selected from—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, and most preferablyselected from —O—P(O)(O⁻)—O— and —O—P(O)(S⁻)—O—.

Thus, the present invention is preferably directed to anantisense-oligonucleotide in form of a gapmer consisting of 10 to 28nucleotides, preferably 11 to 24 nucleotides, more preferably 12 to 20,and still more preferably 13 to 19 or 14 to 18 nucleotides and 1 to 5 ofthese nucleotides at the 5′ terminal end and 1 to 5 nucleotides at the3′ terminal end of the antisense-oligonucleotide are LNA nucleotides andbetween the LNA nucleotides at the 5′ terminal end and the 3′ terminalend a sequence of at least 6, preferably 7 and more preferably 8 DNAnucleotides is present, and the antisense-oligonucleotide is capable ofhybridizing with a region of the gene encoding the TGF-R_(II) or with aregion of the mRNA encoding the TGF-R_(II), wherein theantisense-oligonucleotide is represented by the following sequence5′-N¹¹-TGTTTAGG-N¹²-3′ (Seq. ID No. 10), wherein

N¹¹ represents: GAAGAGCTATTTGGTAG-, AAGAGCTATTTGGTAG-, AGAGCTATTTGGTAG-,GAGCTATTTGGTAG-, AGCTATTTGGTAG-, GCTATTTGGTAG-, CTATTTGGTAG-,TATTTGGTAG-, ATTTGGTAG-, TTTGGTAG-, TTGGTAG-, TGGTAG-, GGTAG-, GTAG-,TAG-, AG- or G-,N¹² represents: -GAGCCGTCTTCAGGAAT, -GAGCCGTCTTCAGGAA, -GAGCCGTCTTCAGGA,-GAGCCGTCTTCAGG, -GAGCCGTCTTCAG, -GAGCCGTCTTCA, -GAGCCGTCTTC,-GAGCCGTCTT, -GAGCCGTCT, -GAGCCGTC, -GAGCCGT, -GAGCCG, -GAGCC, -GAGC,-GAG, -GA, or -G;the LNA nucleotides are selected from β-D-oxy-LNA (b¹), β-D-thio-LNA(b²), α-L-oxy-LNA (b⁴), β-D-ENA (b⁵), β-D-(NH)-LNA (b⁶), β-D-(NCH₃)-LNA(b⁷), β-D-(ONH)-LNA (b⁸) and β-D-(ONCH₃)-LNA (b⁹); and preferably fromβ-D-oxy-LNA (b¹), β-D-thio-LNA (b²), α-L-oxy-LNA (b⁴), β-D-(NH)-LNA(b⁶), and β-D-(NCH₃)-LNA (b⁷); andthe internucleotide linkages are selected from—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—,—S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—, —S—P(O)O⁻)—S—,—O—P(O)(CH₃)—O—, —O—P(O)(OCH₃)—O—, —O—P(O)(NH(CH₃))—O—,—O—P(O)[N(CH₃)₂]—O—, —O—P(O)(BH₃ ⁻)—O—, —O—P(O)(OCH₂CH₂OCH₃)—O—,—O—P(O)(OCH₂CH₂SCH₃)—O—, —O—P(O)(O⁻)—N(CH₃)—, —N(CH₃)—P(O)(O⁻)—O—; andpreferably from —O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—,—S—P(O)(O⁻)—S—, —S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—S—, —S—P(O)(O⁻)—S—;and salts and optical isomers of the antisense-oligonucleotide. Suchpreferred antisense-oligonucleotides may not contain any modified 3′ and5′ terminal end or may not contain any 3′ and 5′ terminal group and mayas modified nucleobase contain 5-methylcytosine and/or 2-aminoadenine.

More preferably N¹ represents: AGAGCTATTTGGTAG-, GAGCTATTTGGTAG-,AGCTATTTGGTAG-, GCTATTTGGTAG-, CTATTTGGTAG-, TATTTGGTAG-, ATTTGGTAG-,TTTGGTAG-, TTGGTAG-, TGGTAG-, GGTAG-, GTAG-, TAG-, AG- or G-; and

N¹² represents: -GAGCCGTCTTCAGGA, -GAGCCGTCTTCAGG, -GAGCCGTCTTCAG,-GAGCCGTCTTCA, -GAGCCGTCTTC, -GAGCCGTCTT, -GAGCCGTCT, -GAGCCGTC,-GAGCCGT, -GAGCCG, -GAGCC, -GAGC, -GAG, -GA, or -G.

Still further preferred, the present invention is directed to anantisense-oligonucleotide in form of a gapmer consisting of 11 to 24nucleotides, more preferably 12 to 20, and still more preferably 13 to19 or 14 to 18 nucleotides and 2 to 5 of these nucleotides at the 5′terminal end and 2 to 5 nucleotides at the 3′ terminal end of theantisense-oligonucleotide are LNA nucleotides and between the LNAnucleotides at the 5′ terminal end and the 3′ terminal end a sequence ofat least 7, preferably at least 8 DNA nucleotides is present, and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the antisense-oligonucleotide is represented by thefollowing sequence 5′-N¹¹-TGTTTAGG-N¹²-3′ (Seq. ID No. 10), wherein

N¹¹ represents: GCTATTTGGTAG-, CTATTTGGTAG-, TATTTGGTAG-, ATTTGGTAG-,TTTGGTAG-, TTGGTAG-, TGGTAG-, GGTAG-, GTAG-, TAG-, AG- or G-; preferablyN¹¹ represents: TATTTGGTAG-, ATTTGGTAG-, TTTGGTAG-, TTGGTAG-, TGGTAG-,GGTAG-, GTAG-, TAG-, AG- or G-; andN¹² represents: -GAGCCGTCTTCA, -GAGCCGTCTTC, -GAGCCGTCTT, -GAGCCGTCT,-GAGCCGTC, -GAGCCGT, -GAGCCG, -GAGCC, -GAGC, -GAG, -GA, or -G;preferably N¹² represents: -GAGCCGTCTT, -GAGCCGTCT, -GAGCCGTC, -GAGCCGT,-GAGCCG, -GAGCC, -GAGC, -GAG, -GA, or -G; and the LNA nucleotides areselected from β-D-oxy-LNA (b¹), β-D-thio-LNA (b²), α-L-oxy-LNA (b⁴),β-D-(NH)-LNA (b⁶), and β-D-(NCH₃)-LNA (b⁷); andthe internucleotide linkages are selected from—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—,—S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—; andpreferably selected from phosphate, phosphorothioate andphosphorodithioate;and salts and optical isomers of the antisense-oligonucleotide. Suchpreferred antisense-oligonucleotides may not contain any modified 3′ and5′ terminal end or may not contain any 3′ and 5′ terminal group and mayas modified nucleobase contain 5-methylcytosine and/or 2-aminoadenine.

Especially preferred are the gapmer antisense-oligonucleotides of Seq.ID No. 369 to Seq. ID No. 403 containing a segment of 2 to 5, preferably2 to 4 and more preferably 3 to 4 LNA units at the 3′ terminus and asegment of 2 to 5, preferably 2 to 4 and more preferably 3 to 4 LNAunits at the 5′ terminus and a segment of at least 6, preferably 7 andmore preferably 8 DNA units between the two segments of LNA units,wherein the LNA units are selected from β-D-oxy-LNA (b¹), β-D-thio-LNA(b²), α-L-oxy-LNA (b⁴), β-D-(NH)-LNA (b⁶), and β-D-(NCH₃)-LNA (b⁷) andthe internucleotide linkages are selected from phosphate,phosphorothioate and phosphorodithioate. Such preferredantisense-oligonucleotides may not contain any modified 3′ and 5′terminal end or may not contain any 3′ and 5′ terminal group and may asmodified nucleobase contain 5-methylcytosine in the LNA units,preferably all the LNA units and/or 2-aminoadenine in some or all DNAunits and/or 5-methylcytosine in some or all DNA units.

Also especially preferred are the gapmer antisense-oligonucleotides ofTable 6 (Seq. ID No. 258a to 270b).

Moreover, the present invention is preferably directed to anantisense-oligonucleotide in form of a gapmer consisting of 10 to 28nucleotides, preferably 11 to 24 nucleotides, more preferably 12 to 20,and still more preferably 13 to 19 or 14 to 18 nucleotides and 1 to 5 ofthese nucleotides at the 5′ terminal end and 1 to 5 nucleotides at the3′ terminal end of the antisense-oligonucleotide are LNA nucleotides andbetween the LNA nucleotides at the 5′ terminal end and the 3′ terminalend a sequence of at least 6, preferably 7 and more preferably 8 DNAnucleotides is present, and the antisense-oligonucleotide is capable ofhybridizing with a region of the gene encoding the TGF-R_(II) or with aregion of the mRNA encoding the TGF-R_(II), wherein theantisense-oligonucleotide is represented by the following sequence5′-N⁵-TTTGGTAG-N⁶-3′ (Seq. ID No. 11), wherein

N⁵ represents: CTGCCCCAGAAGAGCTA-, TGCCCCAGAAGAGCTA-, GCCCCAGAAGAGCTA-,CCCCAGAAGAGCTA-, CCCAGAAGAGCTA-, CCAGAAGAGCTA-, CAGAAGAGCTA-,AGAAGAGCTA-, GAAGAGCTA-, AAGAGCTA-, AGAGCTA-, GAGCTA-, AGCTA-, GCTA-,CTA-, TA-, or A-;N⁶ represents: -TGTTTAGGGAGCCGTCT, -TGTTTAGGGAGCCGTC, -TGTTTAGGGAGCCGT,-TGTTTAGGGAGCCG, -TGTTTAGGGAGCC, -TGTTTAGGGAGC, -TGTTTAGGGAG,-TGTTTAGGGA, -TGTTTAGGG, -TGTTTAGG, -TGTTTAG, -TGTTTA, -TGTTT, -TGTT,-TGT, -TG, or -T;and salts and optical isomers of the antisense-oligonucleotide.

N⁵ and/or N⁶ may also represent any of the further limited lists of 3′and 5′ residues as disclosed herein.

Especially preferred gapmer antisense-oligonucleotides falling undergeneral formula S4:

S4 (Seq. ID No. 11) 5′-N⁵-TTTGGTAG-N⁶-3′are the following:

(Seq. ID No. 404) GAAGAGCTATTTGGTAGT (Seq. ID No. 405)AAGAGCTATTTGGTAGTG (Seq. ID No. 406) AGAGCTATTTGGTAGTGT(Seq. ID No. 407) GAGCTATTTGGTAGTGTT (Seq. ID No. 408)AGCTATTTGGTAGTGTTT (Seq. ID No. 409) GCTATTTGGTAGTGTTTA(Seq. ID No. 410) CTATTTGGTAGTGTTTAG (Seq. ID No. 411)TATTTGGTAGTGTTTAGG (Seq. ID No. 412) ATTTGGTAGTGTTTAGGG(Seq. ID No. 413) AAGAGCTATTTGGTAGT (Seq. ID No. 414) AGAGCTATTTGGTAGTG(Seq. ID No. 415) GAGCTATTTGGTAGTGT (Seq. ID No. 416) AGCTATTTGGTAGTGTT(Seq. ID No. 417) GCTATTTGGTAGTGTTT (Seq. ID No. 418) CTATTTGGTAGTGTTTA(Seq. ID No. 419) TATTTGGTAGTGTTTAG (Seq. ID No. 420) ATTTGGTAGTGTTTAGG(Seq. ID No. 421) AGAGCTATTTGGTAGT (Seq. ID No. 422) GAGCTATTTGGTAGTG(Seq. ID No. 423) AGCTATTTGGTAGTGT (Seq. ID No. 424) GCTATTTGGTAGTGTT(Seq. ID No. 425) CTATTTGGTAGTGTTT (Seq. ID No. 426) TATTTGGTAGTGTTTA(Seq. ID No. 427) ATTTGGTAGTGTTTAG (Seq. ID No. 428) GAGCTATTTGGTAGT(Seq. ID No. 429) AGCTATTTGGTAGTG (Seq. ID No. 430) GCTATTTGGTAGTGT(Seq. ID No. 431) CTATTTGGTAGTGTT (Seq. ID No. 432) TATTTGGTAGTGTTT(Seq. ID No. 433) ATTTGGTAGTGTTTA (Seq. ID No. 434) AGCTATTTGGTAGT(Seq. ID No. 435) GCTATTTGGTAGTG (Seq. ID No. 436) CTATTTGGTAGTGT(Seq. ID No. 437) TATTTGGTAGTGTT (Seq. ID No. 438) ATTTGGTAGTGTTT

The antisense-oligonucleotides of formula S4 in form of gapmers (LNAsegment 1-DNA segment-LNA segment 2) contain an LNA segment at the 5′terminal end consisting of 2 to 5, preferably 2 to 4 LNA units andcontain an LNA segment at the 3′ terminal end consisting of 2 to 5,preferably 2 to 4 LNA units and between the two LNA segments one DNAsegment consisting of 6 to 14, preferably 7 to 12 and more preferably 8to 11 DNA units.

The antisense-oligonucleotides of formula S4 contain the LNA nucleotides(LNA units) as disclosed herein, especially these disclosed in thechapter “Locked Nucleic Acids (LNA®)” and preferably these disclosed inthe chapter “Preferred LNAs”. The LNA units and the DNA units maycomprise standard nucleobases such as adenine (A), cytosine (C), guanine(G), thymine (T) and uracil (U), but may also contain modifiednucleobases as disclosed in the chapter “Nucleobases”. Theantisense-oligonucleotides of formula S4 or the LNA segments and the DNAsegment of the antisense-oligonucleotide may contain any internucleotidelinkage as disclosed herein and especially these disclosed in thechapter “Internucleotide Linkages (IL)”. The antisense-oligonucleotidesof formula S4 may optionally also contain endgroups at the 3′ terminalend and/or the 5′ terminal end and especially these disclosed in thechapter “Terminal groups”.

Experiments have shown that modified nucleobases do not considerablyincrease or change the activity of the inventiveantisense-oligonucleotides in regard to tested neurological andoncological indications. The modified nucleobases 5-methylcytosine or2-aminoadenine have been demonstrated to further increase the activityof the antisense-oligonucleotides of formula S4 especially if5-methylcytosine is used in the LNA nucleotides only or in the LNAnucleotides and in the DNA nucleotides and/or if 2-aminoadenine is usedin the DNA nucleotides and not in the LNA nucleotides.

The preferred gapmer structure of the antisense-oligonucleotides offormula S4 is as follows: 3-8-3, 4-8-2, 2-8-4, 3-8-4, 4-8-3, 4-8-4,3-9-3, 4-9-2, 2-9-4, 4-9-3, 3-9-4, 4-9-4, 3-10-3, 2-10-4, 4-10-2,3-10-4, 4-10-3, 4-10-4, 2-11-4, 4-11-2, 3-11-4, 4-11-3 and still morepreferred: 3-8-3, 3-8-4, 4-8-3, 4-8-4, 3-9-3, 4-9-3, 3-9-4, 4-9-4,3-10-3, 3-10-4, 4-10-3, 4-10-4, 3-11-4, and 4-11-3.

As LNA units for the antisense-oligonucleotides of formula S4 especiallyβ-D-oxy-LNA (b¹), β-D-thio-LNA (b²), α-L-oxy-LNA (b⁴), β-D-ENA (b⁵),β-D-(NH)-LNA (b⁶), β-D-(NCH₃)-LNA (b⁷), β-D-(ONH)-LNA (b⁸) andβ-D-(ONCH₃)-LNA (b⁹) are preferred. Experiments have been shown that allof these LNA units b¹, b², b⁴, b⁵, b⁶, b⁷, b⁸, and b⁹ can be synthesizedwith the required effort and lead to antisense-oligonucleotides ofcomparable stability and activity. However based on the experiments theLNA units b¹, b², b⁴, b⁵, b⁶, and b⁷ are further preferred. Stillfurther preferred are the LNA units b¹, b², b⁴, b⁶, and b⁷, and evenmore preferred are the LNA units b¹ and b⁴ and most preferred also inregard to the complexity of the chemical synthesis is the β-D-oxy-LNA(b¹).

So far no special 3′ terminal group or 5′ terminal group could be foundwhich remarkably had changed or increased the stability or activity foroncological or neurological indications, so that 3′ and 5′ end groupsare possible but not explicitly preferred.

Various internucleotide bridges or internucleotide linkages arepossible. In the formulae disclosed herein the internucleotide linkageIL is represented by -IL′-Y—. Thus, IL=—IL′-Y—═—X″—P(═X′)(X⁻)—Y—,wherein IL is preferably selected form the group consisting of:

—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—,—S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—,—O—P(O)(CH₃)—O—, —O—P(O)(OCH₃)—O—, —O—P(O)(NH(CH₃))—O—,—O—P(O)[N(CH₃)₂]—O—, —O—P(O)(BH₃ ⁻)—O—, —O—P(O)(OCH₂CH₂OCH₃)—O—, —O—P(O)(OCH₂CH₂SCH₃)—O—, —O—P(O)(O⁻)—N(CH₃)—, —N(CH₃)—P(O)(O⁻)—O—. Preferredare the internucleotide linkages IL selected from —O—P(O)(O⁻)—O—,—O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—, —S—P(O)(S⁻)—O—,—O—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—, —O—P(O)(OCH₃)—O—,—O—P(O)(NH(CH₃))—O—, —O—P(O)[N(CH₃)₂]—O—, —O—P(O)(OCH₂CH₂OCH₃)—O—, andmore preferred selected from —O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—,—O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—, —S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—,—O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—, and still more preferred selected from—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, and most preferablyselected from —O—P(O)(O⁻)—O— and —O—P(O)(S⁻)—O—.

Thus, the present invention is preferably directed to anantisense-oligonucleotide in form of a gapmer consisting of 10 to 28nucleotides, preferably 11 to 24 nucleotides, more preferably 12 to 20,and still more preferably 13 to 19 or 14 to 18 nucleotides and 1 to 5 ofthese nucleotides at the 5′ terminal end and 1 to 5 nucleotides at the3′ terminal end of the antisense-oligonucleotide are LNA nucleotides andbetween the LNA nucleotides at the 5′ terminal end and the 3′ terminalend a sequence of at least 6, preferably 7 and more preferably 8 DNAnucleotides is present, and the antisense-oligonucleotide is capable ofhybridizing with a region of the gene encoding the TGF-R_(II) or with aregion of the mRNA encoding the TGF-R_(II), wherein theantisense-oligonucleotide is represented by the following sequence5′-N⁵-TTTGGTAG-N⁶-3′ (Seq. ID No. 11), wherein

N⁵ represents: CTGCCCCAGAAGAGCTA-, TGCCCCAGAAGAGCTA-, GCCCCAGAAGAGCTA-,CCCCAGAAGAGCTA-, CCCAGAAGAGCTA-, CCAGAAGAGCTA-, CAGAAGAGCTA-,AGAAGAGCTA-, GAAGAGCTA-, AAGAGCTA-, AGAGCTA-, GAGCTA-, AGCTA-, GCTA-,CTA-, TA-, or A-; andN⁶ represents: -TGTTTAGGGAGCCGTCT, -TGTTTAGGGAGCCGTC, -TGTTTAGGGAGCCGT,-TGTTTAGGGAGCCG, -TGTTTAGGGAGCC, -TGTTTAGGGAGC, -TGTTTAGGGAG,-TGTTTAGGGA, -TGTTTAGGG, -TGTTTAGG, -TGTTTAG, -TGTTTA, -TGTTT, -TGTT,-TGT, -TG, or -T; andthe LNA nucleotides are selected from β-D-oxy-LNA (b¹), β-D-thio-LNA(b²), α-L-oxy-LNA (b⁴), β-D-ENA (b⁵), β-D-(NH)-LNA (b⁶), β-D-(NCH₃)-LNA(b⁷), β-D-(ONH)-LNA (b⁸) and β-D-(ONCH₃)-LNA (b⁹); and preferably fromβ-D-oxy-LNA (b¹), P-D-thio-LNA (b²), α-L-oxy-LNA (b⁴), β-D-(NH)-LNA(b⁶), and β-D-(NCH₃)-LNA (b⁷); andthe internucleotide linkages are selected from—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—,—S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—, —S—P(O)O⁻)—S—,—O—P(O)(CH₃)—O—, —O—P(O)(OCH₃)—O—, —O—P(O)(NH(CH₃))—O—,—O—P(O)[N(CH₃)₂]—O—, —O—P(O)(BH₃ ⁻)—O—, —O—P(O)(OCH₂CH₂OCH₃)—O—,—O—P(O)(OCH₂CH₂SCH₃)—O—, —O—P(O)(O⁻)—N(CH₃)—, —N(CH₃)—P(O)(O⁻)—O—; andpreferably from —O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—,—S—P(O)(O⁻)—S—, —S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—S—, —S—P(O)(O⁻)—S—;and salts and optical isomers of the antisense-oligonucleotide. Suchpreferred antisense-oligonucleotides may not contain any modified 3′ and5′ terminal end or may not contain any 3′ and 5′ terminal group and mayas modified nucleobase contain 5-methylcytosine and/or 2-aminoadenine.

More preferably N⁵ represents: GCCCCAGAAGAGCTA-, CCCCAGAAGAGCTA-,CCCAGAAGAGCTA-, CCAGAAGAGCTA-, CAGAAGAGCTA-, AGAAGAGCTA-, GAAGAGCTA-,AAGAGCTA-, AGAGCTA-, GAGCTA-, AGCTA-, GCTA-, CTA-, TA-, or A-; and

N⁶ represents: -TGTTTAGGGAGCCGT, -TGTTTAGGGAGCCG, -TGTTTAGGGAGCC,-TGTTTAGGGAGC, -TGTTTAGGGAG, -TGTTTAGGGA, -TGTTTAGGG, -TGTTTAGG,-TGTTTAG, -TGTTTA, -TGTTT, -TGTT, -TGT, -TG, or -T.

Still further preferred, the present invention is directed to anantisense-oligonucleotide in form of a gapmer consisting of 11 to 24nucleotides, more preferably 12 to 20, and still more preferably 13 to19 or 14 to 18 nucleotides and 2 to 5 of these nucleotides at the 5′terminal end and 2 to 5 nucleotides at the 3′ terminal end of theantisense-oligonucleotide are LNA nucleotides and between the LNAnucleotides at the 5′ terminal end and the 3′ terminal end a sequence ofat least 7, preferably at least 8 DNA nucleotides is present, and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the antisense-oligonucleotide is represented by thefollowing sequence 5′-N⁵-TTTGGTAG-N⁶-3′ (Seq. ID No. 11)), wherein

N⁵ represents: CCAGAAGAGCTA-, CAGAAGAGCTA-, AGAAGAGCTA-, GAAGAGCTA-,AAGAGCTA-, AGAGCTA-, GAGCTA-, AGCTA-, GCTA-, CTA-, TA-, or A-;preferably N⁵ represents: AGAAGAGCTA-, GAAGAGCTA-, AAGAGCTA-, AGAGCTA-,GAGCTA-, AGCTA-, GCTA-, CTA-, TA-, or A-; andN⁶ represents: -TGTTTAGGGAGC, -TGTTTAGGGAG, -TGTTTAGGGA, -TGTTTAGGG,-TGTTTAGG, -TGTTTAG, -TGTTTA, -TGTTT, -TGTT, -TGT, -TG, or -T;preferably N⁶ represents: -TGTTTAGGGA, -TGTTTAGGG, -TGTTTAGG, -TGTTTAG,-TGTTTA, -TGTTT, -TGTT, -TGT, -TG, or -T; andthe LNA nucleotides are selected from β-D-oxy-LNA (b¹), β-D-thio-LNA(b²), α-L-oxy-LNA (b⁴), β-D-(NH)-LNA (b⁶), and β-D-(NCH₃)-LNA (b⁷); andthe internucleotide linkages are selected from—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—,—S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—; andpreferably selected from phosphate, phosphorothioate andphosphorodithioate;and salts and optical isomers of the antisense-oligonucleotide. Suchpreferred antisense-oligonucleotides may not contain any modified 3′ and5′ terminal end or may not contain any 3′ and 5′ terminal group and mayas modified nucleobase contain 5-methylcytosine and/or 2-aminoadenine.

Especially preferred are the gapmer antisense-oligonucleotides of Seq.ID No. 404 to Seq. ID No. 438 containing a segment of 2 to 5, preferably2 to 4 and more preferably 3 to 4 LNA units at the 3′ terminus and asegment of 2 to 5, preferably 2 to 4 and more preferably 3 to 4 LNAunits at the 5′ terminus and a segment of at least 6, preferably 7 andmore preferably 8 DNA units between the two segments of LNA units,wherein the LNA units are selected from β-D-oxy-LNA (b¹), β-D-thio-LNA(b²), α-L-oxy-LNA (b⁴), β-D-(NH)-LNA (b⁶), and β-D-(NCH₃)-LNA (b⁷) andthe internucleotide linkages are selected from phosphate,phosphorothioate and phosphorodithioate. Such preferredantisense-oligonucleotides may not contain any modified 3′ and 5′terminal end or may not contain any 3′ and 5′ terminal group and may asmodified nucleobase contain 5-methylcytosine in the LNA units,preferably all the LNA units and/or 2-aminoadenine in some or all DNAunits and/or 5-methylcytosine in some or all DNA units.

Also especially preferred are the gapmer antisense-oligonucleotides ofTable 7 (Seq. ID No. 271a to 283b).

Moreover, the present invention is preferably directed to anantisense-oligonucleotide in form of a gapmer consisting of 10 to 28nucleotides, preferably 11 to 24 nucleotides, more preferably 12 to 20,and still more preferably 13 to 19 or 14 to 18 nucleotides and 1 to 5 ofthese nucleotides at the 5′ terminal end and 1 to 5 nucleotides at the3′ terminal end of the antisense-oligonucleotide are LNA nucleotides andbetween the LNA nucleotides at the 5′ terminal end and the 3′ terminalend a sequence of at least 6, preferably 7 and more preferably 8 DNAnucleotides is present, and the antisense-oligonucleotide is capable ofhybridizing with a region of the gene encoding the TGF-R_(II) or with aregion of the mRNA encoding the TGF-R_(II), wherein theantisense-oligonucleotide is represented by the following sequence5′-N⁷-AATGGACC-N⁸-3′ (Seq. ID No. 100), wherein

N⁷ represents: ATCTTGAATATCTCATG-, TCTTGAATATCTCATG-, CTTGAATATCTCATG-,TTGAATATCTCATG-, TGAATATCTCATG-, GAATATCTCATG-, AATATCTCATG-,ATATCTCATG-, TATCTCATG-, ATCTCATG-, TCTCATG-, CTCATG-, TCATG-, CATG-,ATG-, TG-, or G-;

N⁸

represents: -AGTATTCTAGAAACTCA, -AGTATTCTAGAAACTC, -AGTATTCTAGAAACT,-AGTATTCTAGAAAC, -AGTATTCTAGAAA, -AGTATTCTAGAA, -AGTATTCTAGA,-AGTATTCTAG, -AGTATTCTA, -AGTATTCT, -AGTATTC, -AGTATT, -AGTAT, -AGTA,-AGT, -AG, or -A;and salts and optical isomers of the antisense-oligonucleotide.

N⁷ and/or N⁸ may also represent any of the further limited lists of 3′and 5′ residues as disclosed herein.

Especially preferred gapmer antisense-oligonucleotides falling undergeneral formula S6:

S6 (Seq. ID No. 100) 5′-N⁷-AATGGACC-N⁸-3′are the following:

(Seq. ID No. 439) TATCTCATGAATGGACCA (Seq. ID No. 440)ATCTCATGAATGGACCAG (Seq. ID No. 441) TCTCATGAATGGACCAGT(Seq. ID No. 442) CTCATGAATGGACCAGTA (Seq. ID No. 443)TCATGAATGGACCAGTAT (Seq. ID No. 444) CATGAATGGACCAGTATT(Seq. ID No. 445) ATGAATGGACCAGTATTC (Seq. ID No. 446)TGAATGGACCAGTATTCT (Seq. ID No. 447) GAATGGACCAGTATTCTA(Seq. ID No. 448) ATCTCATGAATGGACCA (Seq. ID No. 449) TCTCATGAATGGACCAG(Seq. ID No. 450) CTCATGAATGGACCAGT (Seq. ID No. 451) TCATGAATGGACCAGTA(Seq. ID No. 452) CATGAATGGACCAGTAT (Seq. ID No. 453) ATGAATGGACCAGTATT(Seq. ID No. 454) TGAATGGACCAGTATTC (Seq. ID No. 455) GAATGGACCAGTATTCT(Seq. ID No. 456) TCTCATGAATGGACCA (Seq. ID No. 457) CTCATGAATGGACCAG(Seq. ID No. 458) TCATGAATGGACCAGT (Seq. ID No. 459) CATGAATGGACCAGTA(Seq. ID No. 460) ATGAATGGACCAGTAT (Seq. ID No. 461) TGAATGGACCAGTATT(Seq. ID No. 462) GAATGGACCAGTATTC (Seq. ID No. 463) CTCATGAATGGACCA(Seq. ID No. 464) TCATGAATGGACCAG (Seq. ID No. 465) CATGAATGGACCAGT(Seq. ID No. 466) ATGAATGGACCAGTA (Seq. ID No. 467) TGAATGGACCAGTAT(Seq. ID No. 468) GAATGGACCAGTATT (Seq. ID No. 469) TCATGAATGGACCA(Seq. ID No. 470) CATGAATGGACCAG (Seq. ID No. 471) ATGAATGGACCAGT(Seq. ID No. 472) TGAATGGACCAGTA (Seq. ID No. 473) GAATGGACCAGTAT

The antisense-oligonucleotides of formula S6 in form of gapmers (LNAsegment 1-DNA segment-LNA segment 2) contain an LNA segment at the 5′terminal end consisting of 2 to 5, preferably 2 to 4 LNA units andcontain an LNA segment at the 3′ terminal end consisting of 2 to 5,preferably 2 to 4 LNA units and between the two LNA segments one DNAsegment consisting of 6 to 14, preferably 7 to 12 and more preferably 8to 11 DNA units.

The antisense-oligonucleotides of formula S6 contain the LNA nucleotides(LNA units) as disclosed herein, especially these disclosed in thechapter “Locked Nucleic Acids (LNA®)” and preferably these disclosed inthe chapter “Preferred LNAs”. The LNA units and the DNA units maycomprise standard nucleobases such as adenine (A), cytosine (C), guanine(G), thymine (T) and uracil (U), but may also contain modifiednucleobases as disclosed in the chapter “Nucleobases”. Theantisense-oligonucleotides of formula S6 or the LNA segments and the DNAsegment of the antisense-oligonucleotide may contain any internucleotidelinkage as disclosed herein and especially these disclosed in thechapter “Internucleotide Linkages (IL)”. The antisense-oligonucleotidesof formula S6 may optionally also contain endgroups at the 3′ terminalend and/or the 5′ terminal end and especially these disclosed in thechapter “Terminal groups”.

Experiments have shown that modified nucleobases do not considerablyincrease or change the activity of the inventiveantisense-oligonucleotides in regard to tested neurological andoncological indications. The modified nucleobases 5-methylcytosine or2-aminoadenine have been demonstrated to further increase the activityof the antisense-oligonucleotides of formula S6 especially if5-methylcytosine is used in the LNA nucleotides only or in the LNAnucleotides and in the DNA nucleotides and/or if 2-aminoadenine is usedin the DNA nucleotides and not in the LNA nucleotides.

The preferred gapmer structure of the antisense-oligonucleotides offormula S6 is as follows: 3-8-3, 4-8-2, 2-8-4, 3-8-4, 4-8-3, 4-8-4,3-9-3, 4-9-2, 2-9-4, 4-9-3, 3-9-4, 4-9-4, 3-10-3, 2-10-4, 4-10-2,3-10-4, 4-10-3, 4-10-4, 2-11-4, 4-11-2, 3-11-4, 4-11-3 and still morepreferred: 3-8-3, 3-8-4, 4-8-3, 4-8-4, 3-9-3, 4-9-3, 3-9-4, 4-9-4,3-10-3, 3-10-4, 4-10-3, 4-10-4, 3-11-4, and 4-11-3.

As LNA units for the antisense-oligonucleotides of formula S6 especiallyβ-D-oxy-LNA (b¹), β-D-thio-LNA (b²), α-L-oxy-LNA (b⁴), β-D-ENA (b⁵),β-D-(NH)-LNA (b⁶), β-D-(NCH₃)-LNA (b⁷), β-D-(ONH)-LNA (b⁸) andβ-D-(ONCH₃)-LNA (b⁹) are preferred. Experiments have been shown that allof these LNA units b¹, b², b⁴, b⁵, b⁶, b⁷, b⁸, and b⁹ can be synthesizedwith the required effort and lead to antisense-oligonucleotides ofcomparable stability and activity. However based on the experiments theLNA units b¹, b², b⁴, b⁵, b⁶, and b⁷ are further preferred. Stillfurther preferred are the LNA units b¹, b², b⁴, b⁶, and b⁷, and evenmore preferred are the LNA units b¹ and b⁴ and most preferred also inregard to the complexity of the chemical synthesis is the β-D-oxy-LNA(b¹).

So far no special 3′ terminal group or 5′ terminal group could be foundwhich remarkably had changed or increased the stability or activity foroncological or neurological indications, so that 3′ and 5′ end groupsare possible but not explicitly preferred.

Various internucleotide bridges or internucleotide linkages arepossible. In the formulae disclosed herein the internucleotide linkageIL is represented by -IL′-Y—. Thus, IL=—IL′-Y—═—X″—P(═X′)(X⁻)—Y—,wherein IL is preferably selected form the group consisting of:

—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—,—S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—,—O—P(O)(CH₃)—O—, —O—P(O)(OCH₃)—O—, —O—P(O)(NH(CH₃))—O—,—O—P(O)[N(CH₃)₂]—O—, —O—P(O)(BH₃ ⁻)—O—, —O—P(O)(OCH₂CH₂OCH₃)—O—,—O—P(O)(OCH₂CH₂SCH₃)—O—, —O—P(O)(O⁻)—N(CH₃)—, —N(CH₃)—P(O)(O⁻)—O—.Preferred are the internucleotide linkages IL selected from—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—,—S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—,—O—P(O)(OCH₃)—O—, —O—P(O)(NH(CH₃))—O—, —O—P(O)[N(CH₃)₂]—O—,—O—P(O)(OCH₂CH₂OCH₃)—O—, and more preferred selected from—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—,—S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—, andstill more preferred selected from —O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—,—O—P(S)(S⁻)—O—, and most preferably selected from —O—P(O)(O⁻)—O— and—O—P(O)(S⁻)—O—.

Thus, the present invention is preferably directed to anantisense-oligonucleotide in form of a gapmer consisting of 10 to 28nucleotides, preferably 11 to 24 nucleotides, more preferably 12 to 20,and still more preferably 13 to 19 or 14 to 18 nucleotides and 1 to 5 ofthese nucleotides at the 5′ terminal end and 1 to 5 nucleotides at the3′ terminal end of the antisense-oligonucleotide are LNA nucleotides andbetween the LNA nucleotides at the 5′ terminal end and the 3′ terminalend a sequence of at least 6, preferably 7 and more preferably 8 DNAnucleotides is present, and the antisense-oligonucleotide is capable ofhybridizing with a region of the gene encoding the TGF-R_(II) or with aregion of the mRNA encoding the TGF-R_(II), wherein theantisense-oligonucleotide is represented by the following sequence5′-N⁷-AATGGACC-N⁸-3′ (Seq. ID No. 100), wherein

N⁷ represents: ATCTTGAATATCTCATG-, TCTTGAATATCTCATG-, CTTGAATATCTCATG-,TTGAATATCTCATG-, TGAATATCTCATG-, GAATATCTCATG-, AATATCTCATG-,ATATCTCATG-, TATCTCATG-, ATCTCATG-, TCTCATG-, CTCATG-, TCATG-, CATG-,ATG-, TG-, or G-; and

N⁸

represents: -AGTATTCTAGAAACTCA, -AGTATTCTAGAAACTC, -AGTATTCTAGAAACT,-AGTATTCTAGAAAC, -AGTATTCTAGAAA, -AGTATTCTAGAA, -AGTATTCTAGA,-AGTATTCTAG, -AGTATTCTA, -AGTATTCT, -AGTATTC, -AGTATT, -AGTAT, -AGTA,-AGT, -AG, or -A; andthe LNA nucleotides are selected from β-D-oxy-LNA (b¹), β-D-thio-LNA(b²), α-L-oxy-LNA (b⁴), β-D-ENA (b⁵), β-D-(NH)-LNA (b⁶), β-D-(NCH₃)-LNA(b⁷), β-D-(ONH)-LNA (b⁸) and β-D-(ONCH₃)-LNA (b⁹); and preferably fromβ-D-oxy-LNA (b¹), β-D-thio-LNA (b²), α-L-oxy-LNA (b⁴), β-D-(NH)-LNA(b⁶), and β-D-(NCH₃)-LNA (b⁷); andthe internucleotide linkages are selected from—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—,—S—P(O)(S⁻)—O—, —O—P(O)(S⁻)—S—, —O—P(O)—, —S—P(O)(O⁻)—S—,—O—P(O)(CH₃)—O—, —O—P(O)(OCH₃)—O—, —O—P(O)(NH(CH₃))—O—,—O—P(O)[N(CH₃)₂]—O—, —O—P(O)(BH₃ ⁻)—O—, —O—P(O)(OCH₂CH₂OCH₃)—O—,—O—P(O)(OCH₂CH₂SCH₃)—O—, —O—P(O)(O⁻)—N(CH₃)—, —N(CH₃)—P(O)(O⁻)—O—; andpreferably from —O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—,—S—P(O)(O⁻)—S—, —S—P(O)(S⁻)—O—, —O—P(O)—, —O—P(S)(S⁻)—O—,—S—P(O)(O⁻)—S—;and salts and optical isomers of the antisense-oligonucleotide. Suchpreferred antisense-oligonucleotides may not contain any modified 3′ and5′ terminal end or may not contain any 3′ and 5′ terminal group and mayas modified nucleobase contain 5-methylcytosine and/or 2-aminoadenine.

More preferably N⁷ represents: CTTGAATATCTCATG-, TTGAATATCTCATG-,TGAATATCTCATG-, GAATATCTCATG-, AATATCTCATG-, ATATCTCATG-, TATCTCATG-,ATCTCATG-, TCTCATG-, CTCATG-, TCATG-, CATG-, ATG-, TG-, or G-; and

N⁸

represents: -AGTATTCTAGAAACT, -AGTATTCTAGAAAC, -AGTATTCTAGAAA, -AGTATTCTAGAA, -AGTATTCTAGA, -AGTATTCTAG, -AGTATTCTA, -AGTATTCT, -AGTATTC, -AGTATT, -AGTAT, -AGTA, -AGT, -AG, or -A.

Still further preferred, the present invention is directed to anantisense-oligonucleotide in form of a gapmer consisting of 11 to 24nucleotides, more preferably 12 to 20, and still more preferably 13 to19 or 14 to 18 nucleotides and 2 to 5 of these nucleotides at the 5′terminal end and 2 to 5 nucleotides at the 3′ terminal end of theantisense-oligonucleotide are LNA nucleotides and between the LNAnucleotides at the 5′ terminal end and the 3′ terminal end a sequence ofat least 7, preferably at least 8 DNA nucleotides is present, and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the antisense-oligonucleotide is represented by thefollowing sequence 5′-N⁷-AATGGACC-N⁸-3′ (Seq. ID No. 100), wherein

N⁷ represents: GAATATCTCATG-, AATATCTCATG-, ATATCTCATG-, TATCTCATG-,ATCTCATG-, TCTCATG-, CTCATG-, TCATG-, CATG-, ATG-, TG-, or G-;preferably N⁷ represents: ATATCTCATG-, TATCTCATG-, ATCTCATG-, TCTCATG-,CTCATG-, TCATG-, CATG-, ATG-, TG-, or G-; and

N⁸

represents: -AGTATTCTAGAA, -AGTATTCTAGA, -AGTATTCTAG, -AGTATTCTA,-AGTATTCT, -AGTATTC, -AGTATT, -AGTAT, -AGTA, -AGT, -AG, or -A;preferably N⁸ represents: -AGTATTCTAG, -AGTATTCTA, -AGTATTCT, -AGTATTC,-AGTATT, -A GTAT, -AGTA, -AGT, -AG, or -A; and the LNA nucleotides areselected from β-D-oxy-LNA (b¹), β-D-thio-LNA (b²), α-L-oxy-LNA (b⁴),β-D-(NH)-LNA (b⁶), and β-D-(NCH₃)-LNA (b⁷); andthe internucleotide linkages are selected from—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—,—S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—; andpreferably selected from phosphate, phosphorothioate andphosphorodithioate;and salts and optical isomers of the antisense-oligonucleotide. Suchpreferred antisense-oligonucleotides may not contain any modified 3′ and5′ terminal end or may not contain any 3′ and 5′ terminal group and mayas modified nucleobase contain 5-methylcytosine and/or 2-aminoadenine.

Especially preferred are the gapmer antisense-oligonucleotides of Seq.ID No. 439 to Seq. ID No. 473 containing a segment of 2 to 5, preferably2 to 4 and more preferably 3 to 4 LNA units at the 3′ terminus and asegment of 2 to 5, preferably 2 to 4 and more preferably 3 to 4 LNAunits at the 5′ terminus and a segment of at least 6, preferably 7 andmore preferably 8 DNA units between the two segments of LNA units,wherein the LNA units are selected from β-D-oxy-LNA (b¹), β-D-thio-LNA(b²), α-L-oxy-LNA (b⁴), β-D-(NH)-LNA (b⁶), and β-D-(NCH₃)-LNA (b⁷) andthe internucleotide linkages are selected from phosphate,phosphorothioate and phosphorodithioate. Such preferredantisense-oligonucleotides may not contain any modified 3′ and 5′terminal end or may not contain any 3′ and 5′ terminal group and may asmodified nucleobase contain 5-methylcytosine in the LNA units,preferably all the LNA units and/or 2-aminoadenine in some or all DNAunits and/or 5-methylcytosine in some or all DNA units.

Also especially preferred are the gapmer antisense-oligonucleotides ofTable 8 (Seq. ID No. 219a to 231b).

Moreover, the present invention is preferably directed to anantisense-oligonucleotide in form of a gapmer consisting of 10 to 28nucleotides, preferably 11 to 24 nucleotides, more preferably 12 to 20,and still more preferably 13 to 19 or 14 to 18 nucleotides and 1 to 5 ofthese nucleotides at the 5′ terminal end and 1 to 5 nucleotides at the3′ terminal end of the antisense-oligonucleotide are LNA nucleotides andbetween the LNA nucleotides at the 5′ terminal end and the 3′ terminalend a sequence of at least 6, preferably 7 and more preferably 8 DNAnucleotides is present, and the antisense-oligonucleotide is capable ofhybridizing with a region of the gene encoding the TGF-R_(II) or with aregion of the mRNA encoding the TGF-R_(II), wherein theantisense-oligonucleotide is represented by the following sequence5′-N⁹-ATTAATAA-N¹⁰-3′ (Seq. ID No. 101), wherein

N⁹ represents: CATATTTATATACAGGC-, ATATTTATATACAGGC-, TATTTATATACAGGC-,ATTTATATACAGGC-, TTTATATACAGGC-, TTATATACAGGC-, TATATACAGGC-,ATATACAGGC-, TATACAGGC-, ATACAGGC-, TACAGGC-, ACAGGC-, CAGGC-, AGGC-,GGC-, GC-, or C-;

N¹⁰

represents: -AGTGCAAATGTTATTGG, -AGTGCAAATGTTATTG, -AGTGCAAATGTTATT,-AGTGCAAATGTTAT, -AGTGCAAATGTTA, -AGTGCAAATGTT, -AGTGCAAATGT,-AGTGCAAATG, -AGTGCAAAT, -AGTGCAAA, -AGTGCAA, -AGTGCA, -AGT GC, -AGTG,-AGT, -AG, or -A;and salts and optical isomers of the antisense-oligonucleotide.

N⁹ and/or N^(o1) may also represent any of the further limited lists of3′ and 5′ residues as disclosed herein.

Especially preferred gapmer antisense-oligonucleotides falling undergeneral formula S7:

S7 (Seq. ID No. 101) 5′-N⁹-ATTAATAA-N¹⁰-3′are the following:

(Seq. ID No. 474) TATACAGGCATTAATAAA (Seq. ID No. 475)ATACAGGCATTAATAAAG (Seq. ID No. 476) TACAGGCATTAATAAAGT(Seq. ID No. 477) ACAGGCATTAATAAAGTG (Seq. ID No. 478)CAGGCATTAATAAAGTGC (Seq. ID No. 479) AGGCATTAATAAAGTGCA(Seq. ID No. 480) GGCATTAATAAAGTGCAA (Seq. ID No. 481)GCATTAATAAAGTGCAAA (Seq. ID No. 482) CATTAATAAAGTGCAAAT(Seq. ID No. 483) ATACAGGCATTAATAAA (Seq. ID No. 484) TACAGGCATTAATAAAG(Seq. ID No. 485) ACAGGCATTAATAAAGT (Seq. ID No. 486) CAGGCATTAATAAAGTG(Seq. ID No. 487) AGGCATTAATAAAGTGC (Seq. ID No. 488) GGCATTAATAAAGTGCA(Seq. ID No. 489) GCATTAATAAAGTGCAA (Seq. ID No. 490) CATTAATAAAGTGCAAA(Seq. ID No. 491) TACAGGCATTAATAAA (Seq. ID No. 492) ACAGGCATTAATAAAG(Seq. ID No. 493) CAGGCATTAATAAAGT (Seq. ID No. 494) AGGCATTAATAAAGTG(Seq. ID No. 495) GGCATTAATAAAGTGC (Seq. ID No. 496) GCATTAATAAAGTGCA(Seq. ID No. 497) CATTAATAAAGTGCAA (Seq. ID No. 498) ACAGGCATTAATAAA(Seq. ID No. 499) CAGGCATTAATAAAG (Seq. ID No. 500) AGGCATTAATAAAGT(Seq. ID No. 501) GGCATTAATAAAGTG (Seq. ID No. 502) GCATTAATAAAGTGC(Seq. ID No. 503) CATTAATAAAGTGCA (Seq. ID No. 504) CAGGCATTAATAAA(Seq. ID No. 505) AGGCATTAATAAAG (Seq. ID No. 506) GGCATTAATAAAGT(Seq. ID No. 507) GCATTAATAAAGTG (Seq. ID No. 508) CATTAATAAAGTGC

The antisense-oligonucleotides of formula S7 in form of gapmers (LNAsegment 1-DNA segment-LNA segment 2) contain an LNA segment at the 5′terminal end consisting of 2 to 5, preferably 2 to 4 LNA units andcontain an LNA segment at the 3′ terminal end consisting of 2 to 5,preferably 2 to 4 LNA units and between the two LNA segments one DNAsegment consisting of 6 to 14, preferably 7 to 12 and more preferably 8to 11 DNA units.

The antisense-oligonucleotides of formula S7 contain the LNA nucleotides(LNA units) as disclosed herein, especially these disclosed in thechapter “Locked Nucleic Acids (LNA®)” and preferably these disclosed inthe chapter “Preferred LNAs”. The LNA units and the DNA units maycomprise standard nucleobases such as adenine (A), cytosine (C), guanine(G), thymine (T) and uracil (U), but may also contain modifiednucleobases as disclosed in the chapter “Nucleobases”. Theantisense-oligonucleotides of formula S7 or the LNA segments and the DNAsegment of the antisense-oligonucleotide may contain any internucleotidelinkage as disclosed herein and especially these disclosed in thechapter “Internucleotide Linkages (IL)”. The antisense-oligonucleotidesof formula S7 may optionally also contain endgroups at the 3′ terminalend and/or the 5′ terminal end and especially these disclosed in thechapter “Terminal groups”.

Experiments have shown that modified nucleobases do not considerablyincrease or change the activity of the inventiveantisense-oligonucleotides in regard to tested neurological andoncological indications. The modified nucleobases 5-methylcytosine or2-aminoadenine have been demonstrated to further increase the activityof the antisense-oligonucleotides of formula S7 especially if5-methylcytosine is used in the LNA nucleotides only or in the LNAnucleotides and in the DNA nucleotides and/or if 2-aminoadenine is usedin the DNA nucleotides and not in the LNA nucleotides.

The preferred gapmer structure of the antisense-oligonucleotides offormula S7 is as follows: 3-8-3, 4-8-2, 2-8-4, 3-8-4, 4-8-3, 4-8-4,3-9-3, 4-9-2, 2-9-4, 4-9-3, 3-9-4, 4-9-4, 3-10-3, 2-10-4, 4-10-2,3-10-4, 4-10-3, 4-10-4, 2-11-4, 4-11-2, 3-11-4, 4-11-3 and still morepreferred: 3-8-3, 3-8-4, 4-8-3, 4-8-4, 3-9-3, 4-9-3, 3-9-4, 4-9-4,3-10-3, 3-10-4, 4-10-3, 4-10-4, 3-11-4, and 4-11-3.

As LNA units for the antisense-oligonucleotides of formula S7 especiallyβ-D-oxy-LNA (b¹), β-D-thio-LNA (b²), α-L-oxy-LNA (b⁴), β-D-ENA (b⁵),β-D-(NH)-LNA (b⁶), β-D-(NCH₃)-LNA (b⁷), β-D-(ONH)-LNA (b⁸) andβ-D-(ONCH₃)-LNA (b⁹) are preferred. Experiments have been shown that allof these LNA units b¹, b², b⁴, b⁵, b⁶, b⁷, b⁸, and b⁹ can be synthesizedwith the required effort and lead to antisense-oligonucleotides ofcomparable stability and activity. However based on the experiments theLNA units b¹, b², b⁴, b⁵, b⁶, and b⁷ are further preferred. Stillfurther preferred are the LNA units b¹, b², b⁴, b⁶, and b⁷, and evenmore preferred are the LNA units b¹ and b⁴ and most preferred also inregard to the complexity of the chemical synthesis is the β-D-oxy-LNA(b¹).

So far no special 3′ terminal group or 5′ terminal group could be foundwhich remarkably had changed or increased the stability or activity foroncological or neurological indications, so that 3′ and 5′ end groupsare possible but not explicitly preferred.

Various internucleotide bridges or internucleotide linkages arepossible. In the formulae disclosed herein the internucleotide linkageIL is represented by -IL′-Y—. Thus, IL=—IL′-Y—═—X″—P(═X′)(X⁻)—Y—,wherein IL is preferably selected form the group consisting of:

—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—,—S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—,—O—P(O)(CH₃)—O—, —O—P(O)(OCH₃)—O—, —O—P(O)(NH(CH₃))—O—,—O—P(O)[N(CH₃)₂]—O—, —O—P(O)(BH₃ ⁻)—O—, —O—P(O)(OCH₂CH₂OCH₃)—O—, —O—P(O)(OCH₂CH₂SCH₃)—O—, —O—P(O)(O⁻)—N(CH₃)—, —N(CH₃)—P(O)(O⁻)—O—. Preferredare the internucleotide linkages IL selected from —O—P(O)(O⁻)—O—,—O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—, —S—P(O)(S⁻)—O—,—O—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—, —O—P(O)(OCH₃)—O—,—O—P(O)(NH(CH₃))—O—, —O—P(O)[N(CH₃)₂]—O—, —O—P(O)(OCH₂CH₂OCH₃)—O—, andmore preferred selected from —O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—,—O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—S—, —S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—S—,—S—P(O)(O⁻)—S—, and still more preferred selected from —O—P(O)(O⁻)—O—,—O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, and most preferably selected from—O—P(O)(O⁻)—O— and —O—P(O)(S⁻)—O—.

Thus, the present invention is preferably directed to anantisense-oligonucleotide in form of a gapmer consisting of 10 to 28nucleotides, preferably 11 to 24 nucleotides, more preferably 12 to 20,and still more preferably 13 to 19 or 14 to 18 nucleotides and 1 to 5 ofthese nucleotides at the 5′ terminal end and 1 to 5 nucleotides at the3′ terminal end of the antisense-oligonucleotide are LNA nucleotides andbetween the LNA nucleotides at the 5′ terminal end and the 3′ terminalend a sequence of at least 6, preferably 7 and more preferably 8 DNAnucleotides is present, and the antisense-oligonucleotide is capable ofhybridizing with a region of the gene encoding the TGF-R_(II) or with aregion of the mRNA encoding the TGF-R_(II), wherein theantisense-oligonucleotide is represented by the following sequence5′-N⁹-ATTAATAA-N¹⁰-3′ (Seq. ID No. 101), wherein

N⁹ represents: CATATTTATATACAGGC-, ATATTTATATACAGGC-, TATTTATATACAGGC-,ATTTATATACAGGC-, TTTATATACAGGC-, TTATATACAGGC-, TATATACAGGC-,ATATACAGGC-, TATACAGGC-, ATACAGGC-, TACAGGC-, ACAGGC-, CAGGC-, AGGC-,GGC-, GC-, or C-;

N¹⁰

represents: -AGTGCAAATGTTATTGG, -AGTGCAAATGTTATTG, -AGTGCAAATGTTATT,-AGTGCAAATGTTAT, -AGTGCAAATGTTA, -AGTGCAAATGTT, -AGTGCAAATGT,-AGTGCAAATG, -AGTGCAAAT, -AGTGCAAA, -AGTGCAA, -AGTGCA, -AGTGC, -AGTG,-AGT, -AG, or -A; andthe LNA nucleotides are selected from β-D-oxy-LNA (b¹), β-D-thio-LNA(b²), α-L-oxy-LNA (b⁴), β-D-ENA (b⁵), β-D-(NH)-LNA (b⁶), β-D-(NCH₃)-LNA(b⁷), β-D-(ONH)-LNA (b⁸) and β-D-(ONCH₃)-LNA (b⁹); and preferably fromβ-D-oxy-LNA (b¹), β-D-thio-LNA (b²), α-L-oxy-LNA (b⁴), β-D-(NH)-LNA(b⁶), and β-D-(NCH₃)-LNA (b⁷); andthe internucleotide linkages are selected from—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—,—S—P(O)(S⁻)—O—, —O—P(O)(S⁻)—S—, —O—P(O)—, —S—P(O)(O⁻)—S—,—O—P(O)(CH₃)—O—, —O—P(O)(OCH₃)—O—, —O—P(O)(NH(CH₃))—O—,—O—P(O)[N(CH₃)₂]—O—, —O—P(O)(BH₃ ⁻)—O—, —O—P(O)(OCH₂CH₂OCH₃)—O—,—O—P(O)(OCH₂CH₂SCH₃)—O—, —O—P(O)(O⁻)—N(CH₃)—, —N(CH₃)—P(O)(O⁻)—O—; andpreferably from —O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—,—S—P(O)(O⁻)—S—, —S—P(O)(S⁻)—O—, —O—P(O)—, —O—P(S)(S⁻)—O—,—S—P(O)(O⁻)—S—;

-   -   and salts and optical isomers of the antisense-oligonucleotide.        Such preferred antisense-oligonucleotides may not contain any        modified 3′ and 5′ terminal end or may not contain any 3′ and 5′        terminal group and may as modified nucleobase contain        5-methylcytosine and/or 2-aminoadenine.

More preferably N⁹ represents: TATTTATATACAGGC-, ATTTATATACAGGC-,TTTATATACAGGC-, TTATATACAGGC-, TATATACAGGC-, ATATACAGGC-, TATACAGGC-,ATACAGGC-, TACAGGC-, ACAGGC-, CAGGC-, AGGC-, GGC-, GC-, or C-; and

N¹⁰

represents: -AGTGCAAATGTTATT, -AGTGCAAATGTTAT, -AGTGCAAATGTTA, -AGTGCAAATGTT, -AGTGCAAATGT, -AGTGCAAATG, -AGTGCAAAT, -AGTGCAAA, -AGTGCAA,-AGTGCA, -AGTGC, -AGTG, -AGT, -AG, or -A.

Still further preferred, the present invention is directed to anantisense-oligonucleotide in form of a gapmer consisting of 11 to 24nucleotides, more preferably 12 to 20, and still more preferably 13 to19 or 14 to 18 nucleotides and 2 to 5 of these nucleotides at the 5′terminal end and 2 to 5 nucleotides at the 3′ terminal end of theantisense-oligonucleotide are LNA nucleotides and between the LNAnucleotides at the 5′ terminal end and the 3′ terminal end a sequence ofat least 7, preferably at least 8 DNA nucleotides is present, and theantisense-oligonucleotide is capable of hybridizing with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the antisense-oligonucleotide is represented by thefollowing sequence 5′-N⁹-ATTAATAA-N¹⁰-3′ (Seq. ID No. 101), wherein

N⁹ represents: TTATATACAGGC-, TATATACAGGC-, ATATACAGGC-, TATACAGGC-,ATACAGGC-, TACAGGC-, ACAGGC-, CAGGC-, AGGC-, GGC-, GC-, or C-;preferably N⁹ represents: ATATACAGGC-, TATACAGGC-, ATACAGGC-, TACAGGC-,ACAGGC-, CAGGC-, AGGC-, GGC-, GC-, or C-; andN¹⁰ represents: -AGTGCAAATGTT, -AGTGCAAATGT, -AGTGCAAATG, -AGTGCAAAT,-AGTGCAAA, -AGTGCAA, -AGTGCA, -AGTGC, -AGTG, -AGT, -AG, or -A;preferably N¹⁰ represents: -AGTGCAAATG, -AGTGCAAAT, -AGTGCAAA, -AGTGCAA,-AGTGCA, -AGTGC, -AGTG, -AGT, -AG, or -A; andthe LNA nucleotides are selected from β-D-oxy-LNA (b¹), β-D-thio-LNA(b²), α-L-oxy-LNA (b⁴), β-D-(NH)-LNA (b⁶), and β-D-(NCH₃)-LNA (b⁷); andthe internucleotide linkages are selected from—O—P(O)(O⁻)—O—, —O—P(O)(S⁻)—O—, —O—P(S)(S⁻)—O—, —S—P(O)(O⁻)—O—,—S—P(O)(S⁻)—O—, —O—P(O)(O⁻)—S—, —O—P(O)(S⁻)—S—, —S—P(O)(O⁻)—S—; andpreferably selected from phosphate, phosphorothioate andphosphorodithioate;and salts and optical isomers of the antisense-oligonucleotide. Suchpreferred antisense-oligonucleotides may not contain any modified 3′ and5′ terminal end or may not contain any 3′ and 5′ terminal group and mayas modified nucleobase contain 5-methylcytosine and/or 2-aminoadenine.

Especially preferred are the gapmer antisense-oligonucleotides of Seq.ID No. 474 to Seq. ID No. 508 containing a segment of 2 to 5, preferably2 to 4 and more preferably 3 to 4 LNA units at the 3′ terminus and asegment of 2 to 5, preferably 2 to 4 and more preferably 3 to 4 LNAunits at the 5′ terminus and a segment of at least 6, preferably 7 andmore preferably 8 DNA units between the two segments of LNA units,wherein the LNA units are selected from β-D-oxy-LNA (b¹), β-D-thio-LNA(b²), α-L-oxy-LNA (b⁴), β-D-(NH)-LNA (b⁶), and β-D-(NCH₃)-LNA (b⁷) andthe internucleotide linkages are selected from phosphate,phosphorothioate and phosphorodithioate. Such preferredantisense-oligonucleotides may not contain any modified 3′ and 5′terminal end or may not contain any 3′ and 5′ terminal group and may asmodified nucleobase contain 5-methylcytosine in the LNA units,preferably all the LNA units and/or 2-aminoadenine in some or all DNAunits and/or 5-methylcytosine in some or all DNA units.

Also especially preferred are the gapmer antisense-oligonucleotides ofTable 9 (Seq. ID No. 284a to 236b).

TABLE 4 Seq ID SP L No. Sequence, 5′-3′ 357 10 232a C*b ¹ sGb ¹sdTsdC*sdAsdTsdAsdGsAb ¹ sC*b ¹ 357 10 232b C*b ¹ Gb ¹dTdC*dAdTdAdGAb ¹C*b ¹ 356 12 233a Tb ¹ sC*b ¹ sGb ¹ sdTsdC*sdAsdTsdAsdGsAb ¹ sC*b ¹ sC*b¹ 356 12 233b Tb ¹ C*b ¹ Gb ¹dTdC*dAdTdAdGAb ¹ C*b ¹ C*b ¹ 356 12 233cTb ¹ sC*b ¹ sGb ¹ sdTsdC*sdAsdTsdAsdGsdAsC*b ¹ sC*b ¹ 356 12 233d Tb ¹sdC*sdGsdTsdC*sdAsdTsdAsdGsdAsC*b ¹ sC*b ¹ 356 12 233e Tb ¹ sC*b ¹sdGsdTsdCsdAsdTsdAsdGsdAsdC*sC*b ¹ 355 13 234a Tb ¹ sC*b ¹ sGb ¹ sTb ¹sdCsdAsdTsdAsdGsdAsC*b ¹ sC*b ¹ sGb ¹ 355 13 234b Tb ¹ C*b ¹ Gb ¹ Tb¹dCdAdUdAdGdAC*b ¹ C*b ¹ Gb ¹ 355 13 234c Tb ¹ sC*b ¹ sGb ¹ sTb ¹sdC*sdAsdTsdAsdGsdAsC*b ¹ sC*b ¹ sGb ¹ 355 13 234d Tb ¹ sC*b ¹ sGb ¹sdTsdC*sdA*sdTsdA*sdGsdA*sdC*sC*b ¹ sGb ¹ 355 13 234e Tb ¹ sC*b ¹sdGsdTsdC*sdAsdTsdA*sdGsdA*sdC*sdCsGb ¹ 355 13 234f Tb ¹sdCsdGsdTsdC*sdA*sdTsdAsdGsAb ¹ sC*b ¹ sC*b ¹ sGb ¹ 354 13 142c C*b ¹sGb ¹ sTb ¹sdCsdAsdTsdAsdGsdAsdCsdCsGb ¹ sAb ¹ 355 14 143i C*b ¹ sTb ¹sC*b ¹ sGb ¹ sdTsdCsdAsdTsdAsdGsAb ¹ sC*b ¹ sC*b ¹ sGb ¹ 355 14 143j C*b⁴ ssTb ⁴ ssC*b ⁴ ssdGssdTssdCssdAssdTssdAssdGssdA*ssC*b ⁴ ssC*b ⁴ ss Gb⁴ 355 14 143h C*b ¹ sTb ¹ sdCsdGsdTsdCsdAsdTsdAsdGsdAsC*b ¹ sC*b ¹ sGb ¹355 14 143k C*b ² ssTb ² ssC*b ² ssdGssdTssdCssdAssdTssdAssdGssdAssC*b ²ssC*b ² ssG b ² 355 14 143m C*b ¹ Tb ¹ C*b ¹ Gb ¹dUsdCsdAsdTsdAsdGsAb ¹C*b ¹ C*b ¹ Gb ¹ 355 14 143n C*b ¹ sTb ¹ sC*b ¹ sGb ¹ sTb ¹sdCsdA*sdTsdA*sdGsdA*sC*b ¹ sC*b ¹ sGb ¹ 355 14 143o C*b ¹ sTb ¹sdCsdGsdUsdCsdAsdUsdAsGb ¹ sAb ¹ sC*b ¹ sC*b ¹ sGb ¹ 355 14 143p C*b ⁶sTb ⁶ sC*b ⁶ sGb ⁶ sdTsdCsdAsdTsdAsdGsdAsC*b ⁶ sC*b ⁶ sGb ⁶ 355 14 143qC*b ⁷ sTb ⁷ sC*b ⁷ sdGsdUsdCsdA*sdUsdA*sdGsdA*sC*b ⁷ sC*b ⁷ sGb ⁷ 355 14143r C*b ⁴ sTb ⁴ sC*b ⁴ sGb ⁴ sdTsdC*sdA*sdTsdAsdGsdAsdC*sC*b ⁴ sGb ⁴355 14 143s C*b ⁴ Tb ⁴ C*b ⁴ Gb ⁴dTdCdAdTdAdGdAdCC*b ⁴ Gb ⁴ 355 14 143tC*b ¹ ssTb ¹ ssC*b ¹ ssdGssdTssdC*ssdAssdTssdAssdGssdAssC*b ¹ ssC*b ¹ ssGb ¹ 355 14 143u C*b ¹ Tb ¹ sdCsdGsdUsdC*sdAsdUsdAsdGsdAsC*b ¹ C*b ¹ Gb¹ 355 14 143v C*b ¹ Tb ¹ sdC*sdGsdTsdC*sdA*sdTsdAsdGsdAsC*b ¹ C*b ¹ Gb ¹355 14 143w C*b ⁶ sTb ⁶ sdC*dGdTdC*dAdTdAdGdAsC*b ⁶ sC*b ⁶ sGb ⁶ 355 14143x C*b ⁷ sTb ⁷ sC*b ⁷ sGb ⁷ sdTsdC*sdAsdTsdAsdGsdAsC*b ⁷ sC*b ⁷ sGb ⁷355 14 143y C*b ⁷ sTb ⁷ sdC*sdGsdTsdCsdAsdUsdAsdGsAb ⁷ sC*b ⁷ sC*b ⁷ sGb⁷ 355 14 143z C*b ¹ sTb ¹ sdC*sdGsdTsdC*sdAsdTsdAsdGsdAsC*b ¹ sC*b ¹ sGb¹ 355 14 143aa C*b ¹ Tb ¹ sdC*sdGsdTsdC*sdAsdTsdAsdGsdAsC*b ¹ C*b ¹ Gb ¹355 14 143ab C*b ¹ sTb ¹ sdC*sdGsdTsdC*sdA*sdTsdAsdGsdA*sC*b ¹ sC*b ¹sGb ¹ 355 14 143ac C*b ¹ sTb ¹ sdC*sdGsdTsdCsdAsdTsdAsdGsdAsC*b ¹ sC*b ¹sGb ¹ 355 14 143ad C*b ¹ Tb ¹dC*dGdTdCdAdTdAdGdAC*b ¹ C*b ¹ Gb ¹ 355 14143ae C*b ¹ sTb ¹ sdC*dGdTdC*dAdTdAdGdAsC*b ¹ sC*b ¹ sGb ¹ 355 14 143af/5SpC3s/C*b ¹ sTb ¹ sdC*dGdTdC*dA*dTdAdGdA*sC*b ¹ sC*b ¹ sGb ¹ 355 14143ag C*b ¹ sTb ¹ sdC*dGdTdC*dA*dTdAdGdA*sC*b ¹ sC*b ¹ sGb ¹/3SpC3s/ 35514 143ah /5SpC3s/C*b ¹ sTb ¹ sdC*dGdTdC*dA*dTdAdGdA*sC*b ¹ sC*b ¹ sGb¹/3SpC3s/ 355 14 143ai C*b ¹ sTb ¹ sdC*sdGsdUsdC*sdA*sdUsdA*sdGsdA*sC*b¹ sC*b ¹ sGb ¹ 355 14 143aj C*b ¹ sTb ¹ sC*b¹sdGsdTsdCsdAsdTsdAsdGsdAsC*b ¹ sC*b ¹ sGb ¹ 356 14 145c Gb ¹ sC*b ¹ sTb¹sdCsdGsdTsdCsdAsdTsdAsdGsAb ¹ sC*b ¹ sC*b ¹ 354 15 235i C*b ¹ sTb ¹sC*b ¹ sGb ¹ sdTdC*dAdTdAdGdAsC*b ¹ sC*b ¹ sGb ¹ sAb ¹ 354 15 235a C*b ¹ssTb ¹ ssdCssdGssdTssdCssdAssdTssdAssdGssdAssdCssdCssdGssAb ¹ 354 15235b C*b ¹ Tb ¹dCdGdTdCdAdTdAdGdAdCdCdGAb ¹ 354 15 235c C*b ¹ sTb ¹sdCsdGsdTsdCsdA*sdUsdAsdGsdAsdCsC*b ¹ sGb ¹ sAb ¹ 354 15 235d C*b ¹ Tb ¹sdCsdGsdTsdCsdAsdTsdAsdGsdAsC*b ¹ C*b ¹ Gb ¹ Ab ¹ 354 15 235e C*b ⁴ sTb⁴ sC*b ⁴ sdGsdTsdCsdAsdTsdAsdGsdAsdCsdCsGb ⁴ sAb ⁴ 354 15 235f C*b ⁶ sTb⁶ sC*b ⁶ sdGdTdCdA*dTdAdGdAdC*sC*b ⁶ sGb ⁶ sAb ⁶ 354 15 235g C*b ¹ sTb ¹sC*b ¹ sGb ¹ sdTsdC*sdAsdTsdAsdGsdAsdC*sdC*sdGsAb ¹ 354 15 235h C*b ¹ssTb ¹ ssdCssdGssdUssdCssdAssdUssdAssdGssdAssdCssdCssGb ¹ ssAb ¹ 355 15144c Gb ¹ sC*b ¹ sTb ¹sdCsdGsdTsdCsdAsdTsdAsdGsdAsC*b ¹ sC*b ¹ sGb ¹ 35416 141c Gb ¹ sC*b ¹ sTb ¹ sC*b ¹ sdGsdTsdC*sdAsdTsdAsdGsdAsC*b ¹ sC*b ¹sGb ¹ sAb ¹ 354 16 141d Gb ¹ C*b ¹ Tb ¹ C*b ¹sdGsdTsdC*sdAsdTsdAsdGsdAsdCsC*b ¹ Gb ¹ Ab ¹ 354 16 141e Gb ⁴ sC*b ⁴ sTb⁴ sC*b ⁴ sdGsdTsdC*sdAsdTsdAsdGsdA*sdC*sdC*sGb ⁴ sAb ⁴ 354 16 141f Gb ¹sdC*sdTsdCsdGsdTsdC*sdA*sdTsdAsdGsdA*sdC*sdC*sdGsAb ¹ 354 16 141g Gb ²sC*b ² sTb ² sdCsdGsdUsdCsdAsdTsdA*sdGsdAsdCsC*b ² sGb ² sAb ² 354 16141h Gb ⁴ ssC*b ⁴ ssTb ⁴ ssdCssdGssdTssdCssdAssdTssdAssdGssdAssC*b ⁴ssC*b ⁴ ssGb ⁴ ssAb ⁴ 354 16 141i Gb ¹ C*b ¹dTdCdGdTdCdA*dTdA*dGdA*dCC*b¹ Gb ¹ Ab ¹ 354 16 141j Gb ¹ sC*b ¹ sTb¹sdCsdGsdTsdCsdAsdTsdAsdGsdAsdCsC*b ¹ sGb ¹ sAb ¹ 351 16 139c C*b ¹ sGb¹ sTb ¹sdCsdAsdTsdAsdGsdAsdCsdCsdGsdAsGb ¹ sC*b ¹ sC*b ¹ 354 17 237a Tb¹ sGb ¹ sC*b ¹ sTb ¹ sC*b ¹ sdGsdTsdC*sdAsdTsdAsdGsAb ¹ sC*b ¹ sC*b ¹sGb ¹ sAb ¹ 354 17 237b Tb ² sGb ² sC*b ² sdTsdGsdTsdC*sdAsdTsdAsdGsAb ²sC*b ² sC*b ² sGb ² sAb ² 354 17 237c Tb ¹ sGb ¹ sC*b ¹ sTb ¹sdC*sdGsdTsdCsdAsdTsdAsdGsdAsdC*sC*b ¹ sGb ¹ sAb ¹ 354 17 237d Tb ¹sdGsdCsdUsdC*sdGsdTsdC*sdAsdUsdAsdGsAb ¹ sC*b ¹ sC*b ¹ sGb ¹ sAb ¹ 35417 237e Tb ¹ sGb ¹ sC*b ¹ sdTsdGsdTsdC*sdA*sdTsdA*sdGsAb ¹ sC*b ¹ sC*b ¹sGb ¹ sAb ¹ 354 17 237f Tb ¹ Gb ¹dC*dTdGdTdC*dAdTdAdGdAC*b ¹ C*b ¹ Gb ¹Ab ¹ 354 17 237g Tb ¹ sdGsdC*sdTsdGsdTsdC*sdAsdTsdAsdGsdAsdC*sC*b ¹ sGb¹ sAb ¹ 354 17 237h Tb ¹ Gb ¹ C*b ¹ Tb ¹ C*b¹dGdTdC*dA*dTdA*dGdA*dC*dC*Gb ¹ Ab ¹ 354 17 237i Tb ¹ ssGb ¹ ssC*b ¹ssTb ¹ ssC*b ¹ ssdGssdTssdCssdAssdTssdAssdGssdAssdC ssC*b ¹ ssGb ¹ ssAb¹ 354 17 237j Tb ⁴ sGb ⁴ sC*b ⁴ sdTdGdTdCdA*dTdA*dGdA*sC*b ⁴ sC*b ⁴ sGb⁴ sAb ⁴ 354 17 237k Tb ⁶ sGb ⁶ sC*b ⁶sdUsdGsdUsdC*sdA*sdUsdA*sdGsdA*sdC*sC*b ⁶ sGb ⁶ sAb ⁶ 354 17 237m Tb ⁷sGb ⁷ sC*b ⁷ sTb ⁷ sdC*dGdTdC*dAdTdAdGdAsC*b ⁷ sC*b ⁷ sGb ⁷ sAb ⁷ 353 18238a Tb ¹ sGb ¹ sC*b ¹ sTb ¹ sC*b ¹ sdGsdTsdC*sdAsdTsdAsdGsdAsC*b ¹ sC*b¹ sGb ¹ sAb ¹ sGb ¹ 353 18 238b Tb ⁷ sGb ⁷ sC*b ⁷ sTb ⁷ sC*b ⁷sdGsdTsdC*sdAsdTsdAsdGsdAsdC*sdC*sdGsdA sGb ⁷ 353 18 238c Tb ¹ sGb ¹sC*b ¹ sTb ¹ sdC*sdGsdTsdCsdAsdTsdAsdGsdAsdC*sC*b ¹ sGb ¹ sAb ¹ sGb ¹353 18 238d Tb ¹ sGb ¹ sdC*sdTsdC*sdGsdTsdCsdAsdTsdAsdGsdAsdC*sdC*sGb ¹sAb ¹ sGb ¹ 353 18 238e Tb ¹ sGb ¹ sC*b ¹ sTb ¹sdC*sdGsdTsdCsdAsdTsdAsdGsdAsdC*sdC*sGb ¹ sAb ¹ sGb ¹ 353 18 238f Tb ¹Gb ¹dC*dUdC*dGdTdC*dAdTdAdGdA*C*b ¹ C*b ¹ Gb ¹ Ab ¹ Gb ¹ 353 18 238g Tb⁴ Gb ⁴ C*b ⁴ Tb ⁴ sdCsdGsdTsdCsdAsdTsdAsdGsdAsC*b ⁴ C*b ⁴ Gb ⁴ Ab ⁴ Gb ⁴353 18 238h Tb ¹ ssGb ¹ ssC*b ¹ssdTssdC*ssdGssdTssdC*ssdAssdTssdA*ssdGssdAssdC* ssdC*ssGb ¹ ssAb ¹ ssGb¹ 353 18 238i Tb ² Gb ² C*b ²dTdCdGdTdC*dAdTdAdGdAC*b ² C*b ² Gb ² Ab ²Gb ² 352 19 239a Tb ¹ sGb ¹ sC*b ¹ sTb ¹ sC*b ¹sdGsdTsdCsdAsdTsdAsdGsdAsdC*sC*b ¹ sGb ¹ sAb ¹ sGb ¹ sC*b ¹ 352 19 239bTb ⁶ Gb ⁶ C*b ⁶ Tb ⁶ C*b ⁶dGdTdC*dAdTdAdGdAdC*C*b ⁶ Gb ⁶ Ab ⁶ Gb ⁶ C*b ⁶352 19 239c Tb ¹ sGb ¹ sC*b ¹ sTb ¹sdC*sdGsdTsdCsdAsdTsdAsdGsdAsdCsdCsdGsAb ¹ sGb ¹ sC*b ¹ 352 19 239d Tb ¹sdGsdCsdTsdCsdGsdTsdCsdAsdTsdAsdGsdA*sdC*sC*b ¹ sGb ¹ sAb ¹ sGb ¹ sC*b ¹352 19 239e Tb ⁴ sGb ⁴ sdCsdUsdCsdGsdUsdCsdAsdTsdAsdGsdA*sdC*sdC*sGb ⁴sAb ⁴ sGb ⁴ sC*b ⁴ 352 19 239f Tb ² ssGb ² ssC*b ² ssTb ² ssC*b ²ssdGssdTssdCssdAssdTssdAssdGssdAssdC ssdCssdGssdAssGb ² ssC*b ² 352 20240a C*b ¹ sTb ¹ sGb ¹ sC*b ¹ sTb ¹ sdC*sdGsdTsdCsdAsdTsdAsdGsdAsdC*sC*b¹ sGb ¹ sAb ¹ sGb ¹ sC*b ¹ 352 20 240b C*b ² sTb ² sGb ²sdC*sdTsdC*sdGsdTsdCsdAsdTsdAsdGsdAsdC*sC*b ² sGb ² sAb ² sGb ² sC*b ²352 20 240c C*b ¹ Tb ¹ Gb ¹dC*dTdC*dGdTdCdAdTdAdGdAdC*dC*Gb ¹ Ab ¹ Gb ¹C*b ¹ 352 20 240d C*b ¹ sdUsdGsdCsdUsdC*sdGsdTsdCsdAsdTsdAsdGsdAsdC*sC*b¹ sGb ¹ sAb ¹ sGb ¹ sC*b ¹ 352 20 240e C*b ⁴ sTb ⁴ sGb ⁴ sC*b ⁴sdTsdCsdGsdTsdCsdAsdTsdAsdGsdAsdCsdCsGb ⁴ sAb ⁴ sGb ⁴ sC*b ⁴ 351 22 241aGb ¹ sC*b ¹ sTb ¹ sGb ¹ sC*b ¹ sdTsdC*sdGsdTsdCsdAsdTsdAsdGsdAsdCsdC*sGb ¹ sAb ¹ sGb ¹ sC*b ¹ sC*b ¹ 351 22 241b Gb ¹ C*b ¹ Tb ¹ Gb ¹ C*b¹dTdC*dGdTdC*dAdTdAdGdAdC*dC*Gb ¹ Ab ¹ Gb ¹ C*b ¹ C*b ¹ 351 22 241c Gb ¹sC*b ¹ sTb ¹ sGb ¹ sC*b ¹ sdTsdCsdGsdTsdCsdAsdTsdAsdGsdAsdCsdCsGb ¹ sAb¹ sGb ¹ sC*b ¹ sC*b ¹ 350 24 242a C*b ¹ sGb ¹ sC*b ¹ sTb ¹ sGb ¹sdCsdTsdCsdGsdTsdCsdAsdTsdAsdGsdAsdCsdC* sdGsAb ¹ sGb ¹ sC*b ¹ sC*b ¹sC*b ¹ 350 24 242b C*b ¹ Gb ¹ C*b ¹ Tb ¹ Gb¹dC*dTdCdGdTdCdAdTdAdGdAdCdC*dGAb ¹ Gb ¹ C*b ¹ C*b ¹ C*b ¹ 349 26 243aC*b ¹ sC*b ¹ sGb ¹ sC*b ¹ sTb ¹ sdGsdC*sdTsdCsdGsdTsdC*sdAsdTsdAsdGsdAsdCsdC*sdGsdAsGb ¹ sC*b ¹ sC*b ¹ sC*b ¹ sC*b ¹ 349 26 243b C*b ¹ C*b ¹ Gb¹ C*b ¹ Tb ¹dGdC*dTdCdGdTdC*dAdTdAdGdAdCdC*dGdAGb ¹ C*b ¹ C*b ¹ C*b ¹C*b ¹ 348 28 244a C*b ¹ sC*b ¹ sC*b ¹ sGb ¹ sC*b ¹sdTsdGsdCsdTsdCsdGsdTsdC*sdAsdTsdAsdGs dAsdC*sdCsdGsdAsdGsC*b ¹ sC*b ¹sC*b ¹ sC*b ¹ sC*b ¹ 348 28 244b C*b ¹ C*b ¹ C*b ¹ Gb ¹ C*b¹dTdGdC*dTdCdGdTdC*dAdTdAdGdAdC*dCdGdAdG C*b ¹ C*b ¹ C*b ¹ C*b ¹ C*b ¹

TABLE 5 Seq ID SP L No. Sequence, 5′-3′ 431 10 245a Tb ¹ sAb ¹sdC*sdGsdCsdGsdTsdC*sC*b ¹ sAb ¹ 431 10 245b Tb ¹ Ab ¹dCdGdC*dGdTdCC*b ¹Ab ¹ 430 12 246a Ab ¹ sTb ¹ sAb ¹ sdC*sdGsdCsdGsdTsdCsC*b ¹ sAb ¹ sC*b ¹430 12 246b Ab ¹ Tb ¹ Ab ¹dCdGdCdGdTdC*C*b ¹ Ab ¹ C*b ¹ 430 12 246c Ab ¹sTb ¹ sAb ¹ sdCsdGsdCsdGsdTsdC*sdC*sAb ¹ sC*b ¹ 430 12 246d Ab ¹ sTb ¹sdA*sdC*sdGsdCsdGsdTsdC*sdC*sdA*sC*b ¹ 430 12 246e Ab ¹sdTsdA*sdC*sdGsdC*sdGsdTsdC*sdC*sAb ¹ sC*b ¹ 430 13 247a Gb ¹ sAb ¹ sTb¹ sAb ¹ sdCsdGsdCsdGsdTsdCsC*b ¹ sAb ¹ sC*b ¹ 430 13 247b Gb ¹ Ab ¹ Tb ¹Ab ¹dCdGdCdGdUdCC*b ¹ Ab ¹ C*b ¹ 430 13 247c Gb ¹ sAb ¹ sTb ¹ sAb ¹sdC*sdGsdCsdGsdTsdC*sC*b ¹ sAb ¹ sC*b ¹ 430 13 247d Gb ¹ sAb ¹ sTb ¹sdA*sdCsdGsdCsdGsdTsdCsdC*sAb ¹ sC*b ¹ 430 13 247e Gb ¹ sAb ¹sdTsdA*sdCsdGsdC*sdGsdTsdCsdC*sdA*sC*b ¹ 430 13 247f Gb ¹sdA*sdTsdA*sdC*sdGsdCsdGsdTsC*b ¹ sC*b ¹ sAb ¹ sC*b ¹ 431 13 153f C*b ¹sGb ¹ sAb ¹sdTsdAsdCsdGsdCsdGsdTsdCsC*b ¹ sAb ¹ 429 14 248a Gb ¹ sAb ¹sTb ¹ sAb ¹ sdC*sdGsdC*sdGsdTsdC*sC*b ¹ sAb ¹ sC*b ¹ sAb ¹ 429 14 248bGb ¹ Ab ¹ Tb ¹ Ab ¹ sdC*sdGsdCsdGsdTsdC*sdC*sAb ¹ C*b ¹ Ab ¹ 429 14 248cGb ⁴ sAb ⁴ sTb ⁴ sAb ⁴ sdC*sdGsdCsdGsdTsdC*sdC*sdA*sC*b ⁴ sAb ⁴ 429 14248d Gb ¹ sdA*sdTsdAsdCsdGsdCsdGsdTsdCsdC*sdA*sdCsAb ¹ 429 14 248e Gb ²sAb ² sTb ² sdA*sdCsdGsdCsdGsdUsdCsdCsAb ² sC*b ² sAb ² 429 14 248f Gb ⁴ssAb ⁴ ssdTssdAssdCssdGssdCssdGssdTssdCssdCssAb ⁴ ssC*b ⁴ ssAb ⁴ 429 14248g Gb ¹ Ab ¹dTdA*dCdGdCdGdTdCC*b ¹ Ab ¹ C*b ¹ Ab ¹ 429 15 152h C*b ¹sGb ¹ sAb ¹ sTb ¹ sdAsdCsdGsdCsdGsdTsdCsdCsAb ¹ sC*b ¹ sAb ¹ 429 15 152iC*b ¹ Gb ¹ Ab ¹ Tb ¹ sdAsdCsdGsdCsdGsdUsdCsdC*sAb ¹ C*b ¹ Ab ¹ 429 15152j C*b ¹ Gb ¹ Ab ¹ Tb ¹ sdA*sdCsdGsdCsdGsdUsdCsdCsAb ¹ C*b ¹ Ab ¹ 42915 152k C*b ⁶ sGb ⁶ sAb ⁶ sTb ⁶ sdAdC*dGdCdGdTdCdC*sAb ⁶ sC*b ⁶ sAb ⁶429 15 152m C*b ¹ sGb ¹ sAb ¹ sTb ¹ sdAsdCsdGsdCsdGsdTsdC*sdC*sAb ¹ sC*b¹ sAb ¹ 429 15 152n C*b ¹ Gb ¹ Ab ¹ Tb ¹ sdAsdC*sdGsdC*sdGsdTsdC*sdC*sAb¹ C*b ¹ Ab ¹ 429 15 152o C*b ¹ sGb ¹ sAb ¹ sTb ¹sdA*sdCsdGsdCsdGsdTsdCsdC*sAb ¹ sC*b ¹ sAb ¹ 429 15 152p C*b ¹ sGb ¹ sAb¹ sTb ¹ sdAsdCsdGsdCsdGsdTsdCsdC*sAb ¹ sC*b ¹ sAb ¹ 429 15 152q C*b ¹ Gb¹ Ab ¹ Tb ¹dAdCdGdC*dGdTdCdC*Ab ¹ C*b ¹ Ab ¹ 429 15 152r C*b ¹ sGb ¹ sAb¹ sTb ¹ sdAdC*dGdC*dGdTdC*dC*sAb ¹ sC*b ¹ sAb ¹ 429 15 152s /5SpC3s/C*b¹ sGb ¹ sAb ¹ sTb ¹ sdAsdC*sdGsdC*sdGsdTsdCsdCsAb ¹ sC*b ¹ sAb ¹ 429 15152t C*b ¹ sGb ¹ sAb ¹ sTb ¹ sdAsdC*sdGsdCsdGsdTsdCsdC*sAb ¹ sC*b ¹ sAb¹ /3SpC3s/ 429 15 152u /5SpC3s/C*b ¹ sGb ¹ sAb ¹ sTb ¹sdAsdC*sdGsdC*sdGsdTsdCsdCsAb ¹ sC*b ¹ sAb ¹/3SpC3s/ 429 15 152v C*b ¹sGb ¹ sAb ¹ sTb ¹ sdA*sdC*sdGsdC*sdGsdUsdC*sdC*sAb ¹ sC*b ¹ sAb ¹ 429 15152w C*b ⁷ sGb ⁷ sAb ⁷ sdTsdAsdCsdGsdC*sdGsdTsdCsC*b ⁷ sAb ⁷ sC*b ⁷ sAb⁷ 429 15 152z C*b ⁷ sGb ⁷ sdAsdUsdAsdCsdGsdC*sdGsdUsdCsC*b ⁷ sAb ⁷ sC*b⁷ sAb ⁷ 429 15 152aa C*b ¹ ssGb ¹ ssAb ¹ssdTssdAssdC*ssdGssdCssdGssdTssdCssdC*ssAb ¹ ssC*b ¹ ssAb ¹ 429 15 152abC*b ⁴ ssGb ⁴ ssAb ⁴ ssdTssdA*ssdCssdGssdCssdGssdTssdCssdCssdA*ss C*b ⁴ssAb ⁴ 429 15 152ac C*b ² ssGb ² ssAb ² ssTb ²ssdAssdCssdGssdCssdGssdTssdCssdCssdAssdCss Ab ² 429 15 152ad C*b ¹ Gb ¹Ab ¹ Tb ¹dAdCdGdCdGdUdCC*b ¹ Ab ¹ C*b ¹ Ab ¹ 429 15 152ae C*b ¹ sGb ¹sAb ¹ sTb ¹ sAb ¹ sdCsdGsdCsdGsdUsdCsdCsAb ¹ sC*b ¹ sAb ¹ 429 15 152afC*b ¹ sGb ¹ sdA*sdTsdA*sdCsdGsdCsdGsdTsC*b ¹ sC*b ¹ sAb ¹ sC*b ¹ sAb ¹429 15 152ag C*b ⁶ sGb ⁶ sAb ⁶ sdTsdAsdCsdGsdCsdGsdTsdCsC*b ⁶ sAb ⁶ sC*b⁶ sAb ⁶ 429 15 152ah C*b ⁷ sGb ⁷ sAb ⁷ sdUsdA*sdCsdGsdCsdGsdUsdCsdCsAb ⁷sC*b ⁷ sAb ⁷ 429 15 152ai C*b ⁴ sGb ⁴ sAb ⁴ sTb ⁴sdA*sdCsdGsdCsdGsdTsdC*sdC*sdA*sC*b ⁴ sAb ⁴ 429 15 152aj C*b ⁴ Gb ⁴ Ab ⁴Tb ⁴dAdCdGdCdGdTdCdCdAC*b ⁴ Ab ⁴ 429 15 152ak C*b ¹ sGb ¹ sAb¹sdTsdAsdCsdGsdCsdGsdTsdCsdCsAb ¹ sC*b ¹ sAb ¹ 428 16 249a C*b ¹ sGb ¹sAb ¹ sTb ¹ sdAdCdGdCdGdTdCdC*sAb ¹ sC*b ¹ sAb ¹ sGb ¹ 428 16 249b C*b ¹ssGb ¹ ssdAssdTssdAssdCssdGssdCssdGssdTssdCssdCssdAssdCssdA ssGb ¹ 42816 249c C*b ¹ Gb ¹dAdTdAdCdGdCdGdTdCdCdAdCdAGb ¹ 428 16 249d C*b ¹ sGb ¹sdAsdUsdAsdC*sdGsdCsdGsdUsdCsdC*sdAsC*b ¹ sAb ¹ sGb ¹ 428 16 249e C*b ¹Gb ¹ sdAsdTsdAsdC*sdGsdC*sdGsdTsdCsdC*sAb ¹ C*b ¹ Ab ¹ Gb ¹ 428 16 249fC*b ⁴ sGb ⁴ sAb ⁴ sdTsdAsdCsdGsdCsdGsdTsdCsdCsdAsdCsAb ⁴ sGb ⁴ 428 16249g C*b ⁶ Gb ⁶ Ab ⁶dTdA*dCdGdCdGdTdC*dCdA*C*b ⁶ Ab ⁶ Gb ⁶ 428 16 249hC*b ¹ sGb ¹ sAb ¹ sTb ¹ sdAsdC*sdGsdCsdGsdTsdCsdC*sdAsdC*sdAsGb ¹ 428 16249i C*b ¹ ssGb ¹ ssdAssdUssdAssdCssdGssdCssdGssdUssdCssdCssdAssdCss Ab¹ ssGb ¹ 428 17 250a Gb ¹ sC*b ¹ sGb ¹ sAb ¹ sTb ¹sdAsdCsdGsdC*sdGsdTsdCsC*b ¹ sAb ¹ sC*b ¹ sAb ¹ sGb ¹ 428 17 250b Gb ¹sC*b ¹ sGb ¹ sAb ¹ sdTsdAsdC*sdGsdC*sdGsdTsdC*sdC*sdAsC*b ¹ sAb ¹ sGb ¹428 17 250c Gb ¹ sdC*sdGsdAsdUsdAsdCsdGsdC*sdGsdUsdCsC*b ¹ sAb ¹ sC*b ¹sAb ¹ sGb ¹ 428 17 250d Gb ¹ sC*b ¹ sGb ¹sdA*sdTsdA*sdC*sdGsdC*sdGsdTsdC*sC*b ¹ sAb ¹ sC*b ¹ sAb ¹ sGb ¹ 428 17250e Gb ¹ C*b ¹dGdAdTdAdCdGdC*dGdTdCdC*Ab ¹ C*b ¹ Ab ¹ Gb ¹ 428 17 250fGb ¹ sdC*sdGsdAsdTsdAsdCsdGsdC*sdGsdTsdCsdCsdAsC*b ¹ sAb ¹ sGb ¹ 428 17250g Gb ² sC*b ² sGb ² sdAsdTsdAsdCsdGsdC*sdGsdTsdC*sC*b ² sAb ² sC*b ²sAb ² sGb ² 428 17 250h Gb ¹ C*b ¹ Gb ¹ Ab ¹ Tb¹dA*dCdGdC*dGdTdC*dCdA*dC*Ab ¹ Gb ¹ 428 17 250i Gb ¹ ssC*b ¹ ssGb ¹ ssAb¹ ssTb ¹ ssdAssdCssdGssdCssdGssdTssdCssdCssdAss C*b ¹ ssAb ¹ ssGb ¹ 42817 250j Gb ⁴ sC*b ⁴ sGb ⁴ sdA*sdTsdA*sdCsdGsdCsdGsdTsdCsdCsAb ⁴ sC*b ⁴sAb ⁴ sGb ⁴ 428 17 250k Gb ⁶ sC*b ⁶ sGb ⁶sdA*sdUsdAsdCsdGsdCsdGsdUsdC*sdCsdA*sC*b ⁶ sAb ⁶ sGb ⁶ 428 17 250m Gb ⁷sC*b ⁷ sGb ⁷ sAb ⁷ sdTdAdCdGdCdGdTdC*dCsAb ⁷ sC*b ⁷ sAb ⁷ sGb ⁷ 427 18251a Gb ¹ sC*b ¹ sGb ¹ sAb ¹ sTb ¹ sdAsdCsdGsdCsdGsdTsdCsdC*sAb ¹ sC*b ¹sAb ¹ sGb ¹ sGb ¹ 427 18 251b Gb ⁷ sC*b ⁷ sGb ⁷ sAb ⁷ sTb ⁷sdAsdC*sdGsdCsdGsdTsdCsdCsdAsdCsdAsdGs Gb ⁷ 427 18 251c Gb ¹ sC*b ¹ sGb¹ sAb ¹ sdTsdAsdC*sdGsdCsdGsdTsdCsdC*sdAsC*b ¹ sAb ¹ sGb ¹ sGb ¹ 427 18251d Gb ¹ sC*b ¹ sGb ¹ sdAsdTsdAsdC*sdGsdC*sdGsdTsdCsdC*sdAsC*b ¹ sAb ¹sGb ¹ sGb ¹ 427 18 251e Gb ¹ sC*b ¹ sGb ¹ sAb ¹sdTsdAsdC*sdGsdCsdGsdTsdCsdC*sdAsdC*sAb ¹ sGb ¹ sGb ¹ 427 18 251f Gb ¹C*b ¹dGdAdUdA*dCdGdCdGdTdC*dC*Ab ¹ C*b ¹ Ab ¹ Gb ¹ Gb ¹ 427 18 251g Gb ⁴C*b ⁴ Gb ⁴ Ab ⁴sdTsdAsdCsdGsdCsdGsdTsdCsdCsAb ⁴ C*b ⁴ Ab ⁴ Gb ⁴ Gb ⁴ 42718 251h Gb ¹ ssC*b ¹ ssGb ¹ssdA*ssdTssdA*ssdCssdGssdCssdGssdTssdCssdCssdA* ssdC*ssAb ¹ ssGb ¹ ssGb¹ 427 18 251i Gb ² C*b ² Gb ²dAdTdAdCdGdC*dGdTdCdC*Ab ² C*b ² Ab ² Gb ²Gb ² 426 19 252a Gb ¹ sC*b ¹ sGb ¹ sAb ¹ sTb ¹sdAsdC*sdGsdCsdGsdTsdCsdCsdAsC*b ¹ sAb ¹ sGb ¹ sGb ¹ sAb ¹ 426 19 252bGb ⁶ C*b ⁶ Gb ⁶ Ab ⁶ Tb ⁶dAdC*dGdCdGdTdCdC*dAC*b ⁶ Ab ⁶ Gb ⁶ Gb ⁶ Ab ⁶426 19 252c Gb ¹ sC*b ¹ sGb ¹ sdAsdTsdAsdCsdGsdCsdGsdTsdCsdCsdAsdC*sAb ¹sGb ¹ sGb ¹ sAb ¹ 426 19 252d Gb ¹sdC*sdGsdA*sdTsdA*sdC*sdGsdCsdGsdTsdCsdCsdA*sC*b ¹ sAb ¹ sGb ¹ sGb ¹ sAb¹ 426 19 252e Gb ⁴ sC*b ⁴ sdGsdAsdUsdAsdCsdGsdCsdGsdUsdCsdCsdAsdC*sAb ⁴sGb ⁴ sGb ⁴ sAb ⁴ 426 19 252f Gb ² ssC*b ² ssGb ² ssAb ² ssTb ²ssdAssdCssdGssdCssdGssdTssdCssdCssdAss dCssdAssdGssGb ² ssAb ² 426 20253a Gb ¹ sGb ¹ sC*b ¹ sGb ¹ sAb ¹ sdTsdAsdCsdGsdCsdGsdTsdC*sdC*sdAsC*b¹ sAb ¹ sGb ¹ sGb ¹ sAb ¹ 426 20 253b Gb ² sGb ² sC*b ²sdGsdAsdTsdAsdC*sdGsdCsdGsdTsdC*sdC*sdAsC*b ² sAb ² sGb ² sGb ² sAb ²426 20 253c Gb ¹ Gb ¹ C*b ¹dGdAdTdAdCdGdCdGdTdCdCdAdC*Ab ¹ Gb ¹ Gb ¹ Ab¹ 426 20 253d Gb ¹ sdGsdCsdGsdAsdTsdAsdCsdGsdC*sdGsdUsdCsdCsdAsC*b ¹ sAb¹ sGb ¹ sGb ¹ sAb ¹ 426 20 253e Gb ⁴ sGb ⁴ sC*b ⁴ sGb ⁴sdAsdTsdAsdCsdGsdCsdGsdTsdCsdCsdAsdCsAb ⁴ sGb ⁴ sGb ⁴ sAb ⁴ 425 22 254aTb ¹ sGb ¹ sGb ¹ sC*Gb ¹ sdAsdTsdAsdCsdGsdCsdGsdTsdCsdCsdAsdC*sAb ¹ sGb¹ sGb ¹ sAb ¹ sC*b ¹ 425 22 254b Tb ¹ Gb ¹ Gb ¹ C*b ¹ Gb¹dAdTdAdC*dGdCdGdTdCdC*dAdCAb ¹ Gb ¹ Gb ¹ Ab ¹ C*b ¹ 425 22 254c Tb ⁶sGb ⁶ sGb ⁶ sC*b ⁶ sdGsdAsdTsdAsdCsdGsdCsdGsdTsdCsdCsdAsdCsdA sdGsGb ⁶sAb ⁶ sC*b ⁶ 424 24 255a C*b ¹ sTb ¹ sGb ¹ sGb ¹ sC*b ¹sdGsdAsdTsdAsdCsdGsdC*sdGsdTsdCsdC*sdA sdCsdAsGb ¹ sGb ¹ sAb ¹ sC*b ¹sGb ¹ 424 24 255b C*b ¹ Tb ¹ Gb ¹ Gb ¹ C*b¹dGdAdTdAdCdGdC*dGdTdCdC*dAdC*dAGb ¹ Gb ¹ Ab ¹ C*b ¹ Gb ¹ 423 26 256a Gb¹ sC*b ¹ sTb ¹ sGb ¹ sGb ¹ sdC*sdGsdAsdTsdAsdCsdGsdCsdGsdTsdCsdCsdAsdCsdAsdGsGb ¹ sAb ¹ sC*b ¹ sGb ¹ sAb ¹ 423 26 256b Gb ¹ C*b ¹ Tb ¹ Gb ¹Gb ¹dC*dGdAdTdAdCdGdCdGdTdCdCdAdC*dAdGGb ¹ Ab ¹ C*b ¹ Gb ¹ Ab ¹ 422 28257a Tb ¹ sGb ¹ sC*b ¹ sTb ¹ sGb ¹sdGsdCsdGsdAsdTsdAsdC*sdGsdC*sdGsdTsdCsdC sdAsdCsdAsdGsGb ¹ sAb ¹ sC*b ¹sGb ¹ sAb ¹ 422 28 257b Tb ¹ Gb ¹ C*b ¹ Tb ¹ Gb¹dGdCdGdAdTdAdCdGdCdGdTdCdC*dAdC*dAdGGb ¹ Ab ¹ C*b ¹ Gb ¹ Ab ¹

TABLE 6 SP L Seq ID No. Sequence, 5′-3′ 2067 10 258a Gb ¹ sTb ¹sdGsdTsdTsdTsdA*sdGsGb ¹ sGb ¹ 2067 10 258b Gb ¹ sTb ¹sdGsdUsdTsdTsdA*sdGsGb ¹ sGb ¹ 2066 12 259a Ab ¹ sGb ¹ sTb ¹sdGsdTsdTsdTsdA*sdGsGb ¹ sGb ¹ sAb ¹ 2066 12 259b Ab ¹ Gb ¹ Tb¹dGdUdUdUdA*dGGb ¹ Gb ¹ Ab ¹ 2066 12 259c Ab ¹ sGb ¹ sTb ¹sdGsdTsdTsdTsdA*sdGsdGsGb ¹ sAb ¹ 2066 12 259d Ab ¹ sGb ¹sdTsdGsdTsdTsdTsdA*sdGsdGsdGsAb ¹ 2066 12 259e Ab ¹sdGsdTsdGsdTsdTsdTsdA*sdGsdGsGb ¹ sAb ¹ 2066 13 260a Tb ¹ sAb ¹ sGb ¹sTb ¹ sdGsdTsdTsdTsdAsdGsGb ¹ sGb ¹ sAb ¹ 2066 13 260b Tb ¹ Ab ¹ Gb ¹ Tb¹dGdUdUdUdAdGGb ¹ Gb ¹ Ab ¹ 2066 13 260c Tb ¹ sAb ¹ sGb ¹ sTb ¹sdGsdTsdTsdTsdA*sdGsGb ¹ sGb ¹ sAb ¹ 2066 13 260d Tb ¹ sAb ¹ sGb ¹sdUsdGsdTsdTsdTsdA*sdGsdGsGb ¹ sAb ¹ 2066 13 260e Tb ¹ sAb ¹sdGsdUsdGsdUsdUsdUsdA*sdGsdGsdGsAb ¹ 2066 13 260f Tb ¹sdA*sdGsdTsdGsdTsdTsdUsdA*sGb ¹ sGb ¹ sGb ¹ sAb ¹ 2065 14 261a Tb ¹ sAb¹ sGb ¹ sTb ¹ sdGsdTsdTsdTsdA*sdGsGb ¹ sGb ¹ sAb ¹ sGb ¹ 2065 14 261b Tb¹ Ab ¹ Gb ¹ Tb ¹ sdGsdTsdTsdTsdA*sdGsdGGb ¹ Ab ¹ Gb ¹ 2065 14 261c Tb ⁴sAb ⁴ sGb ⁴ sTb⁴sdGsdUsdTsdUsdA*sdGsdGsdGsAb ⁴ sGb ⁴ 2065 14 261d Tb ¹sdA*sdGsdUsdGsdTsdTsdUsdA*sdGsdGsdGsdA*sGb ¹ 2065 14 261e Tb ² sAb ² sGb² sdUsdGsdUsdUsdTsdAsdGsdGsGb ² sAb ² sGb ² 2065 14 261f Tb ⁴ sAb ⁴sdGsdTsdGsdTsdTsdTsdAsdGsdGsGb ⁴ sAb ⁴ sGb ⁴ 2065 14 261g Tb ¹ Ab¹dGdTdGdTdTdTdA*dGGb ¹ Gb ¹ Ab ¹ Gb ¹ 2064 15 262a Tb ¹ sAb ¹ sGb ¹ sTb¹ sdGdTdTdTdA*dGdGsGb ¹ sAb ¹ sGb ¹ sC*b ¹ 2064 15 262b Tb ¹ ssAb ¹ssdGssdTssdGssdTssdTssdTssdAssdGssdGssdGssdAssdGssC*b ¹ 2064 15 262c Tb¹ sAb ¹ sdGsdUsdGsdUsdUsdUsdA*sdGsdGsdGsAb ¹ sGb ¹ sC*b ¹ 2064 15 262dTb ¹dAdGdTdGdTdTdTdAdGdGdGdAdGC*b ¹ 2064 15 262e Tb ¹ Ab¹sdGsdUsdGsdUsdTsdUsdAsdGsdGsGb ¹ Ab ¹ Gb ¹ C*b ¹ 2064 15 262f Tb ⁴ sAb⁴ sGb ⁴ sdTsdGsdTsdTsdTsdAsdGsdGsdsdGsdAsGb ⁴ sC*b ⁴ 2064 15 262g Tb ⁶Ab ⁶ Gb ⁶dUdGdTdTdUdAdGdGdGAb ⁶ Gb ⁶ C*b ⁶ 2064 15 262h Tb ¹ sAb ¹ sGb ¹sTb ¹ sdGsdTsdTsdTsdAsdGsdGsdGsdAsdGsC*b ¹ 2064 15 262i Tb ¹ssAb¹ssdGssdTssdGssdUssdUssdUssdAssdGssdGssdGssdAssGb ¹ ssC*b ¹ 2064 16209s Gb ¹ Tb ¹dAsdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsAb ¹ Gb ¹ C*b ¹ 2064 16209t Gb ¹ sTb ¹ sdA*sdGsdTsdGsdTsdTsdTsdA*sdGsdGsdGsAb ¹ sGb ¹ sC*b ¹2064 16 209u Gb ¹ Tb ¹dAdGdTdGdTdTdTdAdGdGdGAb ¹ Gb ¹ C*b ¹ 2064 16 209v/5SpC3s/Gb ¹ sTb ¹ sdAsdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsAb ¹ sGb ¹ sC*b ¹2064 16 209w Gb ¹ sTb ¹ sdAsdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsAb ¹ sGb ¹sC*b ¹/s3SpC3/ 2064 16 209x /5SpC3s/Gb ¹ sTb¹sdAsdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsAb ¹ sGb ¹ sC*b ¹ /3SpC3s/ 2064 16209y Gb ¹ sTb ¹ sdAsdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsAb ¹ sGb ¹ sC*b ¹ 206416 209aa Gb ¹ Tb ¹dA*sdGsdUsdGsdUsdUsdUsdAsdGsdGsdGsAb ¹ Gb ¹ C*b ¹ 206416 209ab Gb ¹ Tb ¹dA*sdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsAb ¹ Gb ¹ C*b ¹ 206416 209ac Gb ⁶ sTb ⁶ sdA*dGdTdGdTdTdTdA*dGdGdGAb ⁶ sGb ⁶ sC*b ⁶ 2064 16209ad Gb ¹ sTb ¹ sdA*sdGsdUsdGsdUsdUsdUsdA*sdGsdGsdGsAb ¹ sGb ¹ sC*b ¹2064 16 209ae Gb ¹ sTb ¹ sdA*sdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsAb ¹ sGb ¹sC*b ¹ 2064 16 209af Gb ¹ sTb ¹ sdAsdGsdTsdGsdTsdTsdTsdA*sdGsdGsdGsAb ¹sGb ¹ sC*b ¹ 2064 16 209ag Gb ¹ Tb ¹dA*dGdTdGdTdTdTdA*dGdGdGAb ¹ Gb ¹C*b ¹ 2064 16 209ah Gb ¹ Tb ¹dAdGdTdGdTdTdTdA*dGdGdGAb ¹ Gb ¹ C*b ¹ 206416 209ai Gb ⁶ sTb ⁶sdA*dGdTdGdTdTdTdAdGdGdGAb ⁶ sGb ⁶ sC*b ⁶ 2064 16209aj Gb ¹ sTb ¹ sdA*sdGsdUsdGsdTsdTsdUsdA*sdGsdGsdGsAb ¹ sGb ¹ sC*b ¹2064 16 209ak Gb ⁷ sTb ⁷ sAb ⁷ sGb ⁷ sdTsdGsdTsdTsdTsdA*sdGsdGsdGsAb ⁷sGb ⁷ sC*b ⁷ 2064 16 209am Gb ⁷ sTb ⁷ sdAsdGsdTsdGsdTsdTsdUsdA*sdGsdGsGb⁷ sAb ⁷ sGb ⁷ sC*b ⁷ 2064 16 209an Gb ¹ ssTb ¹ ssAb ¹ssdGssdTssdGssdTssdTssdTssdA*ssdGssdGssdGssAb ¹ ssGb ¹ ssC*b ¹ 2064 16209ao Gb ⁴ ssTb ⁴ ssAb ⁴ ssdGssdTssdGssdTssdTssdTssdAssdGssdGssdGssdA*ssGb ⁴ ssC*b ⁴ 2064 16 209ap Gb ² ssTb ² ssAb ² ssGb ²ssdTssdGssdTssdTssdTssdAssdGssdGssdGssdAssd GssC*b ² 2064 16 209aq Gb ¹Tb ¹ Ab ¹ Gb ¹dUdGdUdUdUdAdGdGGb ¹ Ab ¹ Gb ¹ C*b ¹ 2064 16 209ar Gb ¹sTb ¹ sAb ¹ sGb ¹ sTb ¹ sdGsdTsdTsdTsdA*sdGsdGsdGsAb ¹ sGb ¹ sC*b ¹ 206416 209as Gb ¹ sTb ¹ sdAsdGsdTsdGsdTsdTsdUsdAsdGsGb ¹ sGb ¹ sAb ¹ sGb ¹sC*b ¹ 2064 16 209at Gb ⁶ sTb ⁶ sAb ⁶ sGb ⁶sdTsdGsdTsdTsdTsdAsdGsdGsdGsAb ⁶ sGb ⁶ sC*b ⁶ 2064 16 209au Gb ⁷ sTb ⁷sAb ⁷ sdGsdUsdGsdTsdTsdTsdA*sdGsdGsdGsAb ⁷ sGb ⁷ sC*b ⁷ 2064 16 209av Gb⁴ sTb ⁴ sAb ⁴ sGb ⁴ sdUsdGsdTsdUsdTsdA*sdGsdGsdGsdA*sGb ⁴ sC*b ⁴ 2064 16209aw Gb ⁴ Tb ⁴ Ab ⁴ Gb ⁴dTdGdTdTdTdAdGdGdGdAGb ⁴ C*b ⁴ 2064 16 209ax Gb¹ sTb ¹ sAb ¹sdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsAb ¹ sGb ¹ sC*b ¹ 2064 16209az Gb ¹ sTb ¹ sAb ¹ sdGsdTsdGsdTsdTsdTsdAsdGsdGsGb ¹ sAb ¹ sGb ¹ sC*b¹ 2064 16 209ba Gb ¹ sTb ¹ sAb ¹ sGb ¹ sdTsdGsdTsdTsdTsdAsdGsdGsGb ¹ sAb¹ sGb ¹ sC*b ¹ 2064 16 209bb Gb ¹ sTb ¹ sAb ¹sdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsdAsGb ¹ sC*b ¹ 2063 17 263a Gb ¹ sTb ¹sAb ¹ sGb ¹ sTb ¹ sdGsdTsdTsdTsdA*sdGsdGsGb ¹ sAb ¹ sGb ¹ sC*b ¹ sC*b ¹2063 17 263b Gb ² sTb ² sAb ² sdGsdTsdGsdTsdTsdTsdA*sdGsdGsGb ² sAb ²sGb ² sC*b ² sC*b ² 2063 17 263c Gb ¹ sTb ¹ sAb ¹ sGb ¹sdTsdGsdTsdTsdTsdA*sdGsdGsdGsAb ¹ sGb ¹ sC*b ¹ sC*b ¹ 2063 17 263d Gb ¹sdUsdA*sdGsdUsdGsdUsdTsdTsdA*sdGsdGsGb ¹ sAb ¹ sGb ¹ sC*b ¹ sC*b ¹ 206317 263e Gb ¹ sTb ¹ sAb ¹ sdGsdTsdGsdUsdTsdTsdA*sdGsdGsGb ¹ sAb ¹ sGb ¹sC*b ¹ sC*b ¹ 2063 17 263f Gb ¹ Tb ¹dA*dGdTdGdTdTdTdA*dGdGdGAb ¹ Gb ¹C*b ¹ C*b ¹ 2063 17 263g Gb ¹sdTsdA*sdGsdTsdGsdTsdTsdTsdA*sdGsdGsdGsdA*sGb ¹ sC*b ¹ sC*b ¹ 2063 17263h Gb ¹ Tb ¹ Ab ¹ Gb ¹ Tb ¹dGdTdUdTdAdGdGdGdA*dGC*b ¹ C*b ¹ 2063 17263i Gb ¹ ssTb ¹ ssAb ¹ ssGb ¹ ssTb ¹ssdGssdTssdTssdTssdAssdGssdGssdGssdAss Gb ¹ ssC*b ¹ ssC*b ¹ 2063 17 263jGb ⁴ Tb ⁴dA*dGdTdGdTdTdTdAdGdGdGdA*Gb ⁴ C*b ⁴ C*b ⁴ 2063 17 263k Gb ⁶sTb ⁶ sAb ⁶ sdGsdTsdGsdUsdUsdTsdAsdGsdGsdGsdA*sGb ⁶ sC*b ⁶ sC*b ⁶ 206317 263m Gb ⁷ sTb ⁷ sAb ⁷ sGb⁷sdTdGdTdTdTdA*dGdGdGsAb ⁷ sGb ⁷ sC*b ⁷ sC*b⁷ 2063 18 264a Gb ¹ sGb ¹ sTb ¹ sAb ¹ sGb ¹ sdTsdGsdTsdTsdTsdA*sdGsdGsGb¹ sAb ¹ sGb ¹ sC*b ¹ sC*b ¹ 2063 18 264b Gb ⁷ sGb ⁷ sTb ⁷ sAb ⁷ sGb ⁷sdTsdGsdTsdTsdTsdAsdGsdGsdGsdAsdGsdC*sC*b ⁷ 2063 18 264c Gb ¹ sGb ¹ sTb¹ sAb ¹ sGb ¹ sdTsdGsdTsdTsdTsdAsdGsdGsdGsdA*sdGsdC*s C*b ¹ 2063 18 264dGb ¹ sGb ¹ sTb ¹ sAb ¹ sGb ¹ sdUsdGsdTsdTsdTsdAsdGsdGsdGsdA*sdGsdC*s C*b¹ 2063 18 264e Gb ¹ sGb ¹ sTb ¹ sAb ¹ sdGsdTsdGsdTsdTsdTsdA*sdGsdGsdGsAb¹ sGb ¹ sC*b ¹ sC*b ¹ 2063 18 264f Gb ¹ sGb ¹ sTb ¹sdA*sdGsdTsdGsdTsdTsdTsdA*sdGsdGsdGsAb ¹ sGb ¹ sC*b ¹ sC*b ¹ 2063 18264g Gb ¹ sGb ¹ sTb ¹ sAb ¹ sdGsdTsdGsdTsdTsdTsdA*sdGsdGsdGsdA*sGb ¹sC*b ¹ sC*b ¹ 2063 18 264h Gb ¹ Gb ¹dUdA*dGdTdGdTdTdTdAdGdGGb ¹ Ab ¹ Gb¹ C*b ¹ C*b ¹ 2063 18 264i Gb ⁴ Gb ⁴ Tb ⁴ Ab⁴dGsdTsdGsdTsdTsdTsdAsdGsdGsGb ⁴ Ab ⁴ Gb ⁴ C*b ⁴ C*b ⁴ 2063 18 264j Gb ¹ssGb ¹ ssTb ¹ ssdA*ssdGssdTssdGssdUssdTssdTssdA*ssdGssdGssdGss dA*ssGb ¹ssC*b ¹ ssC*b ¹ 2063 18 264k Gb ² Gb ² Tb ²dA*dGdTdGdTdTdTdAdGdGGb ² Ab² Gb ² C*b ² C*b ² 2062 19 265a Gb ¹ sGb ¹ sTb ¹ sAb ¹ sGb ¹sdTsdGsdTsdTsdTsdA*sdGsdGsdGsAb ¹ sGb ¹ sC*b ¹ sC*b ¹ sGb ¹ 2062 19 265bGb ⁶ Gb ⁶ Tb ⁶ Ab ⁶ Gb ⁶dTdGdTdTdTdA*dGdGdGAb ⁶ Gb ⁶ C*b ⁶ C*b ⁶ Gb ⁶2062 19 265c Gb ¹ sGb ¹ sTb ¹ sAb ¹sdGsdTsdGsdTsdTsdTsdA*sdGsdGsdGsdA*sdGsC*b ¹ sC*b ¹ sGb ¹ 2062 19 265dGb ¹ sdGsdTsdA*sdGsdUsdGsdTsdUsdTsdA*sdGsdGsdGsAb ¹ sGb ¹ sC*b ¹ sC*b ¹sGb ¹ 2062 19 265e Gb ⁴ sGb ⁴sdUsdAsdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsdA*sGb ⁴ sC*b ⁴ sC*b ⁴ sGb ⁴ 206219 265f Gb ² ssGb ² ssTb ² ssAb ² ssGb ²ssdTssdGssdTssdTssdTssdAssdGssdGssdGss dAssdGssdCssC*b ² ssGb ² 2062 20266a Tb ¹ sGb ¹ sGb ¹ sTb ¹ sAb ¹ sdGsdTsdGsdTsdTsdTsdA*sdGsdGsdGsAb ¹sGb ¹ sC*b ¹ sC*b ¹ sGb ¹ 2062 20 266b Tb ² sGb ² sGb ²sdTsdA*sdGsdTsdGsdTsdTsdTsdA*sdGsdGsdGsAb ² sGb ² sC*b ² sC*b ² sGb ²2062 20 266c Gb ¹ Gb ¹ Tb ¹dA*dGdTdGdTdTdTdA*dGdGdGdA*Gb ¹ C*b ¹ C*b ¹Gb ¹ 2062 20 266d Tb ¹ sdGsdGsdUsdA*sdGsdTsdGsdTsdUsdTsdA*sdGsdGsdGsAb ¹sGb ¹ sC*b ¹ sC*b ¹ sGb ¹ 2062 20 266e Tb ⁴ sGb ⁴ sGb ⁴sTb⁴sdAsdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsdAsGb ⁴ sC*b ⁴ sC*b ⁴ sGb ⁴ 206122 267a Tb ¹ sTb ¹ sGb ¹ sGb ¹ sTb ¹sdAsdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsdA*sGb ¹ sC*b ¹ sC*b ¹ sGb ¹ sTb ¹2061 22 267b Tb ¹ Tb ¹ Gb ¹ Gb ¹ Tb ¹dA*dGdTdGdTdTdTdAdGdGdGdA*Gb ¹ C*b¹ C*b ¹ Gb ¹ Tb ¹ 2061 22 267c Tb ⁶ sTb ⁶ sGb ⁶sdGsdTsdAsdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsdAsGb ⁶ sC*b ⁶ sC*b ⁶ sGb ⁶ sTb⁶ 2060 24 268a Tb ¹ sTb ¹ sTb ¹ sGb ¹ sGb ¹sdTsdA*sdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsdA sdGC*b ¹ sC*b ¹ sGb ¹ sTb ¹sC*b ¹ 2060 24 268b Tb ¹ Tb ¹ Tb ¹ Gb ¹ Gb¹dTdA*dGdTdGdTdTdTdAdGdGdGdA*dGC*b ¹ C*b ¹ Gb ¹ Tb ¹ C*b ¹ 2059 26 269aAb ¹ sTb ¹ sTb ¹ sTb ¹ sGb ¹ sdGsdTsdAsdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsdAsdGsdC*sC*b ¹ sGb ¹ sTb ¹ sC*b ¹ sTb ¹ 2059 26 269b Ab ¹ Tb ¹ Tb ¹ Tb¹ Gb ¹dGdTdAdGdTdGdTdTdTdAdGdGdGdAdGdC*C*b ¹ Gb ¹ Tb ¹ C*b ¹ Tb ¹ 205828 270a Tb ¹ sAb ¹ sTb ¹ sTb ¹ sTb ¹sdGsdGsdTsdAsdGsdTsdGsdTsdTsdTsdAsdGsdG sdGsdAsdGsdC*sdCsGb ¹ sTb ¹ sC*b¹ sTb ¹ sTb ¹ 2058 28 270b Tb ¹ Ab ¹ Tb ¹ Tb ¹ Tb¹dGdGdTdAdGdTdGdTdTdTdAdGdGdGdAdGdC*dC*Gb ¹ Tb ¹ C*b ¹ Tb ¹ Tb ¹

TABLE 7 SP L Seq ID No. Sequence, 5′-3′ 2075 10 271a Ab ¹ sTb ¹sdTsdTsdGsdGsdTsdA*sGb ¹ sTb ¹ 2075 10 271b Ab ¹ Tb ¹dTdTdGdGdTdA*Gb ¹Tb ¹ 2074 12 272a Tb ¹ sAb ¹ sTb ¹ sdTsdTsdGsdGsdTsdA*sGb ¹ sTb ¹ sGb ¹2074 12 272b Tb ¹ Ab ¹ Tb ¹dTdTdGdGdTdA*Gb ¹ Tb ¹ Gb ¹ 2074 12 272c Tb ¹sAb ¹ sTb ¹ sdTsdTsdGsdGsdTsdA*sdGsTb ¹ sGb ¹ 2074 12 272d Tb ¹ sAb ¹sdTsdTsdTsdGsdGsdTsdA*sdGsdUsGb ¹ 2074 12 272e Tb ¹sdAsdTsdUsdTsdGsdGsdUsdA*sdGsTb ¹ sGb ¹ 2073 13 273a Tb ¹ sAb ¹ sTb ¹sdTsdTsdGsdGsdTsdAsGb ¹ sTb ¹ sGb ¹ sTb ¹ 2073 13 273b Tb ¹ Ab ¹ Tb¹dUdUdGdGdUdAGb ¹ Tb ¹ Gb ¹ Tb ¹ 2073 13 273c Tb ¹ sAb ¹ sTb ¹sdTsdTsdGsdGsdTsdA*sGb ¹ sTb ¹ sGb ¹ sTb ¹ 2073 13 273d Tb ¹ sAb ¹ sTb ¹sdTsdTsdGsdGsdTsdA*sdGsdUsGb ¹ sTb ¹ 2073 13 273e Tb ¹ sAb ¹sdUsdUsdUsdGsdGsdUsdA*sdGsdUsdGsTb ¹ 2073 13 273f Tb ¹sdA*sdTsdTsdUsdGsdGsdTsdA*sGb ¹ sTb ¹ sGb ¹ sTb ¹ 2073 14 274a C*b ¹ Tb¹ Ab ¹ sdUsdTsdTsdGsdGsdTsdA*sGb ¹ Tb ¹ Gb ¹ Tb ¹ 2073 14 274b C*b ⁴ sTb⁴ sAb ⁴ sTb ⁴ sdTsdTsdGsdGsdTsdA*sdGsdUsGb ⁴ sTb ⁴ 2073 14 274c C*b ¹sdUsdA*sdTsdTsdTsdGsdGsdTsdAsdGsdTsdGsTb ¹ 2073 14 274d C*b ² sTb ² sAb² sdTsdTsdUsdGsdGsdTsdA*sdGsTb ² sGb ² sTb ² 2073 14 274e C*b ⁴ ssTb ⁴ssdAssdTssdTssdTssdGssdGssdTssdAssdGssTb ⁴ ssGb ⁴ ssTb ⁴ 2073 14 274fC*b ¹ Tb ¹ Ab ¹dTdTdTdGdGdTdA*Gb ¹ Tb ¹ Gb ¹ Tb ¹ 2073 14 274g C*b ¹ sTb¹ sAb ¹ sTb ¹ sdTsdTsdGsdGsdTsdA*sGb ¹ sTb ¹ sGb ¹ sTb ¹ 2072 15 275aC*b ¹ sTb ¹ sAb ¹ sTb ¹ sdTsdTsdGsdGsdTsdA*sdGsdTsdGsdTsTb ¹ 2072 15275b C*b ¹ sTb ¹ sdA*sdUsdTsdUsdGsdGsdTsdAsdGsdUsGb ¹ sTb ¹ sTb ¹ 207215 275c C*b ⁴ sTb ⁴ sAb ⁴ sdTsdTsdTsdGsdGsdTsdAsdGsdTsdGsTb ⁴ sTb ⁴ 207215 275d C*b ¹ ssTb ¹ssdAssdTssdTssdTssdGssdGssdTssdAssdGssdTssdGssdTssTb ¹ 2072 15 275e C*b¹ ssTb ¹ ssdAssdUssdTssdTssdGssdGssdTssdAssdGssdUssdGssTb ¹ ssTb ¹ 207215 275f C*b ¹ sTb ¹ sAb ¹ sTb ¹ sdTdTdGdGdTdA*dGsTb ¹ sGb ¹ sTb ¹ sTb ¹2072 15 275g C*b ¹ Tb ¹ sdAsdTsdTsdTsdGsdGsdTsdA*sdGsTb ¹ Gb ¹ Tb ¹ Tb ¹2072 15 275h C*b ⁶ Tb ⁶ Ab ⁶dUdTdTdGdGdTdA*dGdUGb ⁶ Tb ⁶ Tb ⁶ 2072 15275i C*b ¹dTdAdTdTdTdGdGdTdAdGdTdGdTTb ¹ 2072 16 210o Gb ¹ C*b ¹ Tb ¹ Ab¹dTsdTsdTsdGsdGsdTsdAsdGsdTsGb ¹ Tb ¹ Tb ¹ 2072 16 210p Gb ¹ sC*b ¹ sTb¹ sAb ¹ sdTsdTsdTsdGsdGsdTsdA*sdGsdTsGb ¹ sTb ¹ sTb ¹ 2072 16 210q Gb ¹sC*b ¹ sTb ¹ sAb ¹ sdTsdTsdTsdGsdGsdTsdAsdGsdTsGb ¹ sTb ¹ sTb ¹ 2072 16210r Gb ¹ C*b ¹ Tb ¹ Ab ¹dTdTdTdGdGdTdA*dGdTGb ¹ Tb ¹ Tb ¹ 2072 16 210sGb ¹ sC*b ¹ sTb ¹ sAb ¹ sdUsdUsdTdGsdGsdTsdA*sdGsdTsGb ¹ sTb ¹ sTb ¹2072 16 210t Gb ¹ sC*b ¹ sTb ¹ sAb ¹ sdUsdTsdTsdGsdGsdTsdAsdGsdUsGb ¹sTb ¹ sTb ¹ 2072 16 210u Gb ¹ sC*b ¹ sTb ¹ sAb ¹sdUsdUsdUsdGsdGsdUsdA*sdGsdUsGb ¹ sTb ¹ sTb ¹ 2072 16 210v /5SpC3s/Gb ¹sC*b ¹ sTb ¹ sAb ¹ sdTsdTsdTsdGsdGsdTsdAsdGsdTsGb ¹ sTb ¹ sTb ¹ 2072 16210w Gb ¹ sC*b ¹ sTb ¹ sAb ¹ sdTsdTsdTsdGsdGsdTsdAsdGsdTsGb ¹ sTb ¹ sTb¹ /3SpC3s/ 2072 16 210x /5SpC3s/ Gb ¹ sC*b ¹ sTb ¹ sAb ¹sdTsdTsdTsdGsdGsdTsdAsdGsdTsGb ¹ sTb ¹ sTb ¹ /3SpC3s/ 2072 16 210y Gb ¹C*b ¹ Tb ¹ Ab ¹ sdTsdTsdTsdGsdGsdTsdA*sdGsdTsGb ¹ Tb ¹ Tb ¹ 2072 16 210zGb ¹ C*b ¹ Tb ¹ Ab ¹ sdUsdTsdTsdGsdGsdUsdA*sdGsdTsGb ¹ Tb ¹ Tb ¹ 2072 16210aa Gb ¹ sC*b ¹ sTb ¹ sAb ¹ sdTdTdTdGdGdTdA*dGdTsGb ¹ sTb ¹ sTb ¹ 207216 210ab Gb ⁶ sC*b ⁶ sTb ⁶ sAb ⁶ sdTdTdTdGdGdTdA*dGdTsGb ⁶ sTb ⁶ sTb ⁶2072 16 210ac Gb ⁶ sC*b ⁶ sTb ⁶ sdAsdTsdTsdTsdGsdGsdTsdAsdGsTb ⁶ sGb ⁶sTb ⁶ sTb ⁶ 2072 16 210ad Gb ⁷ sC*b ⁷ sTb ⁷sdA*sdTsdTsdTsdGsdGsdTsdA*sdGsTb ⁷ sGb ⁷ sTb ⁷ sTb ⁷ 2072 16 210ae Gb ⁷sC*b ⁷ sdUsdAsdTsdTsdUsdGsdGsdUsdA*sdGsTb ⁷ sGb ⁷ sTb ⁷ sTb ⁷ 2072 16210af Gb ¹ ssC*b ¹ ssTb ¹ ssdAssdTssdTssdTssdGssdGssdTssdA*ssdGssdTssGb¹ ssTb ¹ ssTb ¹ 2072 16 210ag Gb ⁴ ssC*b ⁴ ssTb ⁴ssdA*ssdTssdTssdTssdGssdGssdTssdAssdGssdTssdGss Tb ⁴ ssTb ⁴ 2072 16210ah Gb ² ssC*b ² ssTb ²ssAb²ssdTssdTssdTssdGssdGssdTssdAssdGssdTssdGss dTssTb ² 2072 16 210aiGb ¹ C*b ¹ Tb ¹ Ab ¹dUsdTsdTsdGsdGsdTsdAsdGsTb ¹ Gb ¹ Tb ¹ Tb ¹ 2072 16210aj Gb ⁴ C*b ⁴ Tb ⁴ Ab ⁴dTsdTsdTsdGsdGsdTsdAsdGsdTdGTb ⁴ Tb ⁴ 2072 16210ak Gb ¹ sC*b ¹ sTb ¹ sAb ¹ sTb ¹ sdTsdTsdGsdGsdTsdA*sdGsdTsGb ¹ sTb ¹sTb ¹ 2072 16 210am Gb ⁴ sC*b ⁴ sTb ⁴ sAb ⁴sdTsdTsdUsdGsdGsdTsdA*sdGsdTsdGsTb ⁴ sTb ⁴ 2072 16 210an Gb ⁷ sC*b ⁷ sTb⁷ sdA*sdTsdTsdUsdGsdGsdTsdA*sdGsdTsGb ⁷ sTb ⁷ sTb ⁷ 2072 16 210ao Gb ¹sC*b ¹ sdUsdAsdUsdUsdTsdGsdGsdUsdAsGb ¹ sTb ¹ sGb ¹ sTb ¹ sTb ¹ 2072 16210ap Gb ¹ sC*b ¹ sTb ¹sdAsdTsdTsdTsdGsdGsdTsdAsdGsdTsGb ¹ sTb ¹ sTb ¹2072 16 210aq Gb ¹ sC*b ¹ sTb ¹ sdAsdTsdTsdTsdGsdGsdTsdAsdGsdTsdGsTb ¹sTb ¹ 2071 17 276a Gb ¹ sC*b ¹ sTb ¹ sAb ¹ sTb ¹sdTsdTsdGsdGsdTsdA*sdGsTb ¹ sGb ¹ sTb ¹ sTb ¹ sTb ¹ 2071 17 276b Gb ²sC*b ² sTb ² sdAsdTsdTsdTsdGsdGsdTsdA*sdGsTb ² sGb ² sTb ² sTb ² sTb ²2071 17 276c Gb ¹ sC*b ¹ sTb ¹ sdAsdTsdTsdTsdGsdGsdTsdA*sdGsdTsGb ¹ sTb¹ sTb ¹ sTb ¹ 2071 17 276d Gb ² sdC*sdTsdAsdTsdTsdTsdGsdGsdTsdAsdGsTb ²sGb ² sTb ² sTb ² sTb ² 2071 17 276e Gb ⁶ sC*b ⁶ sTb ⁶sdA*sdUsdUsdUsdGsdGsdUsdA*sdGsdUsdGsTb ⁶ sTb ⁶ sTb ⁶ 2071 17 276f Gb ¹sdC*sdTsdA*sdUsdUsdUsdGsdGsdUsdAsdGsdUsdGsTb ¹ sTb ¹ sTb ¹ 2071 17 276gGb ¹ C*b ¹dTdA*dTdTdTdGdGdTdA*dGdTGb ¹ Tb ¹ Tb ¹ Tb ¹ 2071 17 276h Gb ⁴C*b ⁴ Tb ⁴ Ab ⁴dTdTdTdGdGdTdA*dGdTdGTb ⁴ Tb ⁴ Tb ⁴ 2071 17 276i Gb ¹ C*b¹ Tb ¹ Ab ¹ Tb ¹dUdTdGdGdTdA*dGdTdGdUTb ¹ Tb ¹ 2071 17 276j Gb ¹ ssC*b ¹ssTb ¹ ssAb ¹ ssTb ¹ ssdTssdTssdGssdGssdTssdAssdGssdTssdGss Tb ¹ ssTb ¹ssTb ¹ 2071 17 276k Gb ⁷ sC*b ⁷ sTb ⁷ sAb ⁷ sdTdTdTdGdGdTdA*dGdTsGb ⁷sTb ⁷ sTb ⁷ sTb ⁷ 2071 18 277a Ab ¹ sGb ¹ sC*b ¹ sTb ¹ sAb ¹sdTsdTsdTsdGsdGsdTsdA*sdGsTb ¹ sGb ¹ sTb ¹ sTb ¹ sTb ¹ 2071 18 277b Ab ⁷sGb ⁷ sC*b ⁷ sTb ⁷ sAb ⁷ sdTsdTsdTsdGsdGsdTsdA*sdGsdTsdGsdTsdTsTb ⁷ 207118 277c Ab ¹ sGb ¹ sdC*sdTsdAsdTsdTsdTsdGsdGsdTsdAsdGsTb ¹ sGb ¹ sTb ¹sTb ¹ sTb ¹ 2071 18 277d Ab ¹ sGb ¹sdC*sdTsdAsdTsdTsdTsdGsdGsdTsdA*sdGsTb ¹ sGb ¹ sTb ¹ sTb ¹ sTb ¹ 2071 18277e Ab ¹ Gb ¹dC*dTdAdUdTdTdGdGdTdA*dGTb ¹ Gb ¹ Tb ¹ Tb ¹ Tb ¹ 2071 18277f Ab ² Gb ² C*b ²dTdAdTdTdTdGdGdTdA*dGTb ² Gb ² Tb ² Tb ² Tb ² 207118 277g Ab ¹ sGb ¹ sC*b ¹ sTb ¹ sdA*sdTsdTsdTsdGsdGsdTsdA*sdGsdTsGb ¹sTb ¹ sTb ¹ sTb ¹ 2071 18 277h Ab ¹ sGb ¹ sC*b ¹ sTb ¹sdA*sdTsdTsdTsdGsdGsdTsdAsdGsdTsdGsTb ¹ sTb ¹ sTb ¹ 2071 18 277i Ab ¹sGb ¹ sC*b ¹ sdTsdA*sdTsdTsdTsdGsdGsdTsdA*sdGsdTsGb ¹ sTb ¹ sTb ¹ sTb ¹2071 18 277j Ab ⁴ Gb ⁴ C*b ⁴ Tb ⁴ sdAsdTsdTsdTsdGsdGsdTsdAsdGsTb ⁴ Gb ⁴Tb ⁴ Tb ⁴ Tb ⁴ 2071 18 277k Ab ¹ ssGb ¹ ssC*b ¹ssdTssdA*ssdTssdTssdTssdGssdGssdTssdA*ssdGssdUssd GssTb ¹ ssTb ¹ ssTb ¹2070 19 278a Ab ¹ sGb ¹ sC*b ¹ sTb ¹ sAb ¹sdTsdTsdTsdGsdGsdTsdA*sdGsdTsGb ¹ sTb ¹ sTb ¹ sTb ¹ sAb ¹ 2070 19 278bAb ² ssGb ² ssC*b ² ssTb ² ssAb ² ssdTssdTssdTssdGssdGssdTssdAssdGssdTssdGssdTssdTssTb ² ssAb ² 2070 19 278c Ab ¹sdGsdC*sdTsdAsdTsdTsdTsdGsdGsdTsdA*sdGsdTsGb ¹ sTb ¹ sTb ¹ sTb ¹ sAb ¹2070 19 278d Ab ¹ sdGsdC*sdTsdAsdTsdTsdTsdGsdGsdUsdA*sdGsdUsGb ¹ sTb ¹sTb ¹ sTb ¹ sAb ¹ 2070 19 278e Ab ¹ sGb ¹ sC*b ¹sdTsdAsdTsdTsdTsdGsdGsdTsdA*sdGsdTsdGsTb ¹ sTb ¹ sTb ¹ sAb ¹ 2070 19278f Ab ⁴ sGb ⁴ sdC*sdTsdAsdTsdTsdTsdGsdGsdTsdAsdGsdTsdGsTb ⁴ sTb ⁴ sTb⁴ sAb ⁴ 2070 19 278g Ab ⁶ Gb ⁶ C*b ⁶ Tb ⁶ Ab ⁶dTdTdTdGdGdTdA*dGdTGb ⁶ Tb⁶ Tb ⁶ Tb ⁶ Ab ⁶ 2070 20 279a Gb ¹ sAb ¹ sGb ¹ sC*b ¹ sTb ¹sdAsdTsdTsdTsdGsdGsdTsdA*sdGsdTsGb ¹ sTb ¹ sTb ¹ sTb ¹ sAb ¹ 2070 20279b Gb ² sAb ² sGb ² sdC*sdTsdAsdTsdTsdTsdGsdGsdTsdAsdGsdTsGb ² sTb ²sTb ² sTb ² sAb ² 2070 20 279c Gb ¹sdAsdGsdC*sdUsdAsdTsdTsdTsdGsdGsdTsdA*sdGsdTsGb ¹ sTb ¹ sTb ¹ sTb ¹ sAb¹ 2070 20 279d Gb ⁴ sAb ⁴ sGb ⁴ sC*b ⁴sdTsdAsdTsdTsdTsdGsdGsdTsdAsdGsdTsdGsTb ⁴ sTb ⁴ sTb ⁴ sAb ⁴ 2070 20 279eGb ¹ Ab ¹ Gb ¹dC*dTdAdTdTdTdGdGdTdAdGdTdGTb ¹ Tb ¹ Tb ¹ Ab ¹ 2069 22280a Ab ¹ sGb ¹ sAb ¹ sGb ¹ sC*b ¹sdTsdAsdTsdTsdTsdGsdGsdTsdAsdGsdTsdGsTb ¹ sTb ¹ sTb ¹ sAb ¹ sGb ¹ 206922 280b Ab ¹ Gb ¹ Ab ¹ Gb ¹ C*b ¹dTdAdTdTdTdGdGdTdAdGdTdGTb ¹ Tb ¹ Tb ¹Ab ¹ Gb ¹ 2069 22 280c Ab ¹ sGb ¹ sAb ¹ sGb ¹sdC*sdTsdAsdTsdTsdTsdGsdGsdTsdAsdGsdTsdGsTb ¹ sTb ¹ sTb ¹ sAb ¹ sGb ¹2069 22 280d Ab ⁶ sGb ⁶ sAb ⁶ sGb ⁶sdC*sdTsdAsdTsdTsdTsdGsdGsdTsdA*sdGsdTsdGsdTsd TsTb ⁶ sAb ⁶ sGb ⁶ 206824 281a Ab ¹ sAb ¹ sGb ¹ sAb ¹ sGb ¹sdC*sdTsdAsdTsdTsdTsdGsdGsdTsdAsdGsdTsdGs dTsTb ¹ sTb ¹ sAb ¹ sGb ¹ sGb¹ 2068 24 281b Ab ¹ Ab ¹ Gb ¹ Ab ¹ Gb ¹dC*dTdAdTdTdTdGdGdTdAdGdTdGdTTb ¹Tb ¹ Ab ¹ Gb ¹ Gb ¹ 2067 26 282a Gb ¹ sAb ¹ sAb ¹ sGb ¹ sAb ¹sdGsdC*sdTsdAsdTsdTsdTsdGsdGsdTsdAsdGsdTs dGsdTsdTsTb ¹ sAb ¹ sGb ¹ sGb¹ sGb ¹ 2067 26 282b Gb ¹ Ab ¹ Ab ¹ Gb ¹ Ab¹dGdC*dTdAdTdTdTdGdGdTdAdGdTdGdTdTTb ¹ Ab ¹ Gb ¹ Gb ¹ Gb ¹ 2066 28 283aAb ¹ sGb ¹ sAb ¹ sAb ¹ sGb ¹ sdAsdGsdC*sdTsdAsdTsdTsdTsdGsdGsdTsdAsdGsdTsdGsdTsdTsdTsAb ¹ sGb ¹ sGb ¹ sGb ¹ sAb ¹ 2066 28 283b Ab ¹ Gb ¹ Ab ¹Ab ¹ Gb ¹dAdGdC*dTdAdTdTdTdGdGdTdAdGdTdGdTdTdTAb ¹ Gb ¹ Gb ¹ Gb ¹ Ab ¹

TABLE 8 SP L Seq ID No. Sequence, 5′-3′ 4220 10 219a Gb ¹ sAb ¹sdAsdTsdGsdGsdAsdCsC*b ¹ sAb ¹ 4220 10 219b Gb ¹ Ab ¹dAdTdGdGdAdCC*b ¹Ab ¹ 4219 12 220a Tb ¹ sGb ¹ sAb ¹ sdAsdTsdGsdGsdAsdCsC*b ¹ sAb ¹ sGb ¹4219 12 220b Tb ¹ Gb ¹ Ab ¹dAdTdGdGdAdCC*b ¹ Ab ¹ Gb ¹ 4219 12 220c Tb ¹sGb ¹ sAb ¹ sdAsdTsdGsdGsdAsdCsdC*sAb ¹ sGb ¹ 4219 12 220d Tb ¹sdGsdA*sdAsdTsdGsdGsdAsdC*sdCsAb ¹ sGb ¹ 4219 12 220e Tb ¹ sGb ¹sdA*sdA*sdTsdGsdGsdA*sdC*sdC*sdAsGb ¹ 4218 13 221a Tb ¹ sGb ¹ sAb ¹ sAb¹ sdTsdGsdGsdAsdCsdCsAb ¹ sGb ¹ sTb ¹ 4218 13 221b Tb ¹ Gb ¹ Ab ¹ Ab¹dUdGdGdAdCdCAb ¹ Gb ¹ Tb ¹ 4218 13 221c Tb ¹ sGb ¹ sAb ¹ sAb ¹sdTsdGsdGsdAsdCsdC*sAb ¹ sGb ¹ sTb ¹ 4218 13 221d Tb ¹ sGb ¹ sAb ¹sdAsdTsdGsdGsdA*sdCsdC*sdAsGb ¹ sTb ¹ 4218 13 221e Tb ¹ sGb ¹sdA*sdAsdTsdGsdGsdAsdC*sdCsdAsdGsTb ¹ 4218 13 221f Tb ¹sdGsdAsdA*sdTsdGsdGsdAsdCsC*b ¹ sAb ¹ sGb ¹ sTb ¹ 4218 14 222a Ab ¹ sTb¹ sGb ¹ sAb ¹ sdAsdTsdGsdGsdAsdCsC*b ¹ sAb ¹ sGb ¹ sTb ¹ 4218 14 222b Ab¹ Tb ¹ Gb ¹ Ab ¹dAsdTsdGsdGsdAsdCsdC*sAb ¹ Gb ¹ Tb ¹ 4218 14 222c Ab ¹Tb ¹dGdA*dAdTdGdGdA*dCC*b ¹ Ab ¹ Gb ¹ Tb ¹ 4218 14 222d Ab ⁴ sTb ⁴ sGb ⁴sdA*sdAsdTsdGsdGsdAsdCsdC*sAbsGb ⁴ sTb ⁴ 4218 14 222e Ab ¹sdTsdGsdA*sdA*sdTsdGsdGsdA*sdC*sdC*sdA*sdGsTb ¹ 4218 14 222f Ab ² sTb ²sGb ² sdA*sdAsdUsdGsdGsdAsdCsdCsAb ² sGb ² sTb ² 4218 14 222g Ab ⁴ ssTb⁴ ssdGssdAssdAssdTssdGssdGssdAssdCssdCssAb ⁴ ssGb ⁴ ssTb ⁴ 4217 15 223aAb ¹ sTb ¹ sGb ¹ sAb ¹ sdAdTdGdGdAdCdC*sAb ¹ sGb ¹ sTb ¹ sAb ¹ 4217 15223b Ab ¹ ssTb ¹ ssdGssdAssdAssdTssdGssdGssdAssdCssdCssdAssdGssdTssAb ¹4217 15 223c Ab ¹dTdGdAdAdTdGdGdAdCdCdAdGdTAb ¹ 4217 15 223d Ab ¹ sTb ¹sdGsdAsdAsdUsdGsdGsdA*sdCsdCsdAsGb ¹ sTb ¹ sAb ¹ 4217 15 223e Ab ⁶ Tb ⁶Gb ⁶dA*dAdTdGdGdAdCdC*dAGb ⁶ Tb ⁶ Ab ⁶ 4217 15 223f Ab ¹ Tb¹dGsdAsdAsdTsdGsdGsdAsdC*sdC*sAb ¹ Gb ¹ Tb ¹ Ab ¹ 4217 15 223g Ab ⁴ sTb⁴ sGb ⁴ sdAsdAsdTsdGsdGsdAsdCsdCsdAsdGsTb ⁴ sAb ⁴ 4217 15 223h Ab ¹ sTb¹ sGb ¹ sAb¹sdAsdTsdGsdGsdAsdC*sdC*sdAsdGsdTsAb ¹ 4217 15 223i Ab ¹ ssTb¹ ssdGssdAssdAssdUssdGssdGssdA*ssdCssdCssdAssdGssTb ¹ ssAb ¹ 4217 16218y C*b ² sAb ² sTb ² sdGsdAsdAsdTsdGsdGsdAsdCsdCsAb ² sGb ² sTb ² sAb² 4217 16 218z C*b ¹ sAb ¹ sTb ¹ sdGsdAsdAsdTsdGsdGsdAsdC*sdC*sAb ¹ sGb¹ sTb ¹ sAb ¹ 4217 16 218aa C*b ¹ ssAb ¹ ssTb ¹ssdGssdAssdAssdTssdGssdGssdAssdCssdCssAb ¹ ssGb ¹ ssTb ¹ ssAb ¹ 4217 16218ab C*b ¹ Ab ¹ Tb ¹dGsdAsdAsdUsdGsdGsdAsdC*sdC*sAb ¹ Gb ¹ Tb ¹ Ab ¹4217 16 218ac C*b ¹ Ab ¹ Tb ¹dGsdA*sdA*sdTsdGsdGsdA*sdCsdCsAb¹Gb¹Tb¹Ab¹4217 16 218ad C*b ⁶ sAb ⁶ sTb ⁶ sdGdAdAdTdGdGdAdCdCAb ⁶ sGb ⁶ sTb ⁶ sAb⁶ 4217 16 218ae C*b ⁷ sAb ⁷ sTb ⁷ sGb ⁷ sdAsdAsdTsdGsdGsdAsdCsdCsdAsGb ⁷sTb ⁷ sAb ⁷ 4217 16 218af C*bs ¹ Ab ¹ sdUsdGsdAsdAsdUsdGsdGsdUsdCsdCsAb¹ sGb ¹ sTb ¹ sAb ¹ 4217 16 218b C*b ¹ sAb ¹ sTb¹sdGsdAsdAsdTsdGsdGsdAsdCsdCsAb ¹ sGb ¹ sTb ¹ sAb ¹ 4217 16 218m C*b ¹sAb ¹ sTb ¹ sdGsdAsdAsdTsdGsdGsdAsdC*sdC*sAb ¹ sGb ¹ sTb ¹ sAb ¹ 4217 16218n C*b ¹ Ab ¹ Tb ¹dGsdAsdAsdTsdGsdGsdAsdC*sdC*sAb ¹ Gb ¹ Tb ¹ Ab ¹4217 16 218o C*b ¹ sAb ¹ sTb ¹ sdGsdA*sdA*sdTsdGsdGsdA*sdCsdCsAb ¹ sGb ¹sTb ¹ sAb ¹ 4217 16 218p C*b ¹ sAb ¹ sTb ¹sdGsdA*sdA*sdTsdGsdGsdA*sdC*sdC*sAb ¹ sGb ¹ sTb ¹ sAb ¹ 4217 16 218q C*b¹ sAb ¹ sTb ¹ sdGsdAsdAsdTsdGsdGsdAsdC*sdCsAb ¹ sGb ¹ sTb ¹ sAb ¹ 421716 218c C*b ¹ sAb ¹ sTb ¹ sdGsdAsdAsdTsdGsdGsdAsdCsdC*sAb ¹ sGb ¹ sTb ¹sAb ¹ 4217 16 218r C*b ¹ Ab ¹ Tb ¹dGdAdAdTdGdGdAdCdCAb ¹ Gb ¹ Tb ¹ Ab ¹4217 16 218s C*b ¹ sAb ¹ sTb¹sdGdAdAdTdGdGdAdC*sdC*sAb ¹ sGb ¹ sTb ¹ sAb¹ 4217 16 218t /5SpC3s/C*b ¹ sAb ¹ sTb ¹ sdGsdAsdAsdTsdGsdGsdAsdCsdCsAb¹ sGb ¹ sTb ¹ sAb ¹ 4217 16 218u C*b ¹ sAb ¹ sTb ¹sdGsdAsdAsdTsdGsdGsdAsdCsdCsAb ¹ sGb ¹ sTb ¹ sAb ¹ /3SpC3s/ 4217 16 218v/5SpC3s/C*b ¹ sAb ¹ sTb ¹ sdGsdAsdAsdTsdGsdGsdAsdCsdCsAb ¹ sGb ¹ sTb ¹sAb ¹/3SpC3s/ 4217 16 218ag C*b ¹ sAb ¹ sTb ¹sdGsdA*sdA*sdUsdGsdGsdA*sdCsdCsAb ¹ sGb ¹ sTb ¹ sAb ¹ 4217 16 218ah C*b⁴ ssAb ⁴ ssTb ⁴ ssdGssdA*ssdA*ssdTssdGssdGssdA*ssdCssdCssdAssdGss Tb ⁴ssAb ⁴ 4217 16 218ai C*b ² ssAb ² ssTb ² ssGb ²ssdAssdAssdTssdGssdGssdAssdCssdCssdAssdGss dTssAb ² 4217 16 218aj C*b ¹Ab ¹ Tb ¹ Gb ¹dAdAdUdGdGdAdCdCAb ¹ Gb ¹ Tb ¹ Ab ¹ 4217 16 218ak C*b ¹sAb ¹ sTb ¹ sGb ¹ sAb ¹ sdA*sdUsdGsdGsdAsdCsdCsdA*sGb ¹ sTb ¹ sAb ¹ 421716 218am C*b ¹ sAb ¹ sdUsdGsdAsdAsdUsdGsdGsdAsdCsC*b ¹ sAb ¹ sGb ¹ sTb ¹sAb ¹ 4217 16 218an C*b ⁶ sAb ⁶ sTb ⁶ sGb ⁶sdAsdAsdTsdGsdGsdAsdCsdCsdAsGb ⁶ sTb ⁶ sAb ⁶ 4217 16 218ao C*b ⁷ sAb ⁷sTb ⁷ sdGsdA*sdA*sdUsdGsdGsdAsdCsdCsdA*sGb ⁷ sTb ⁷ sAb ⁷ 4217 16 218apC*b ⁴ sAb ⁴ sTb ⁴ sGb ⁴ sdA*sdAsdTsdGsdGsdAsdCsdC*sdAsdGsTb ⁴ sAb ⁴ 421716 218aq C*b ⁴ Ab ⁴ Tb ⁴ Gb ⁴dAdAdTdGdGdAdCdCdAdGTb ⁴ Ab ⁴ 4217 16 218arC*b ¹ sAb ¹ sTb ¹sdGsdAsdAsdTsdGsdGsdAsdCsdCsdAsGb ¹ sTb ¹ sAb ¹ 4216 17224a C*b ¹ sAb ¹ sTb ¹ sGb ¹ sAb ¹ sdAsdTsdGsdGsdAsdCsdCsAb ¹ sGb ¹ sTb¹ sAb ¹ sTb ¹ 4216 17 224b C*b ² sAb ² sTb ²sdGsdAsdAsdTsdGsdGsdAsdCsdCsAb ² sGb ² sTb ² sAb ² sTb ² 4216 17 224cC*b ¹ sAb ¹ sTb ¹ sGb ¹ sdAsdAsdTsdGsdGsdAsdCsdCsdAsdGsTb ¹ sAb ¹ sTb ¹4216 17 224d C*b ¹ sdAsdUsdGsdAsdAsdUsdGsdGsdAsdC*sdC*sAb ¹ sGb ¹ sTb ¹sAb ¹ sTb ¹ 4216 17 224e C*b ¹ sAb ¹ sTb ¹sdGsdA*sdA*sdTsdGsdGsdA*sdC*sdC*sAb ¹ sGb ¹ sTb ¹ sAb ¹ sTb ¹ 4216 17224f C*b ¹ Ab ¹dTdGdAdAdTdGdGdAdCdCdAGb ¹ Tb ¹ Ab ¹ Tb ¹ 4216 17 224gC*b ¹ sdAsdTsdGsdAsdAsdTsdGsdGsdAsdCsdCsdAsdGsTb ¹ sAb ¹ sTb ¹ 4216 17224h C*b ¹ Ab ¹ Tb ¹ Gb ¹ Ab ¹dA*dTdGdGdA*dC*dC*dAdGdTAb ¹ Tb ¹ 4216 17224i C*b ¹ ssAb ¹ ssTb ¹ ssGb ¹ ssAb ¹ssdAssdTssdGssdGssdAssdCssdCssdAssdGss Tb ¹ ssAb ¹ ssTb ¹ 4216 17 224jC*b ⁴ Ab ⁴ Tb ⁴dGdA*dA*dTdGdGdA*dCdCdAGb ⁴ Tb ⁴ Ab ⁴ Tb ⁴ 4216 17 224kC*b ⁶ sAb ⁶ sTb ⁶ sdGsdA*sdA*sdUsdGsdGsdA*sdC*sdC*sdAsdGsTb ⁶ sAb ⁶ sTb⁶ 4216 17 224m C*b ⁷ sAb ⁷ sTb ⁷ sGb ⁷ sdAdAdTdGdGdAdC*dC*dAsGb ⁷ sTb ⁷sAb ⁷ sTb ⁷ 4216 18 225a Tb ¹ sC*b ¹ sAb ¹ sTb ¹ sGb ¹sdAsdAsdTsdGsdGsdAsdCsdCsAb ¹ sGb ¹ sTb ¹ sAb ¹ sTb ¹ 4216 18 225b Tb ⁷sC*b ⁷ sAb ⁷ sTb ⁷ sGb ⁷ sdAsdAsdTsdGsdGsdAsdCsdCsdAsdGsdTsdAsTb ⁷ 421618 225c Tb ¹ sC*b ¹ sAb ¹ sTb ¹ sdGsdAsdAsdTsdGsdGsdAsdC*sdC*sdAsGb ¹sTb ¹ sAb ¹ sTb ¹ 4216 18 225d Tb ¹ sC*b ¹ sAb ¹sdTsdGsdAsdAsdTsdGsdGsdAsdCsdCsdAsGb ¹ sTb ¹ sAb ¹ sTb ¹ 4216 18 225e Tb¹ sC*b ¹ sAb ¹ sTb ¹ sdGsdAsdAsdTsdGsdGsdAsdCsdCsdAsdGsTb ¹ sAb ¹ sTb ¹4216 18 225f Tb ¹ C*b ¹dA*dTdGdAdAdUdGdGdAdCdC*Ab ¹ Gb ¹ Tb ¹ Ab ¹ Tb ¹4216 18 225g Tb ⁴ C*b ⁴ Ab ⁴ Tb ⁴ sdGsdAsdAsdTsdGsdGsdAsdCsdCsAb ⁴ Gb ⁴Tb ⁴ Ab ⁴ Tb ⁴ 4216 18 225h Tb ¹ ssC*b ¹ ssAb ¹ssdTssdGssdA*ssdA*ssdTssdGssdGssdAssdCssdC*ssdA* ssdGssTb ¹ ssAb ¹ ssTb¹ 4216 18 225i Tb ² C*b ² Ab ²dTdGdAdAdTdGdGdAdC*dC*Ab ² Gb ² Tb ² Ab ²Tb ² 4215 19 226a Tb ¹ sC*b ¹ sAb ¹ sTb ¹ sGb ¹sdAsdAsdTsdGsdGsdAsdCsdCsdAsGb ¹ sTb ¹ sAb ¹ sTb ¹ sTb ¹ 4215 19 226b Tb⁶ C*b ⁶ Ab ⁶ Tb ⁶ Gb ⁶dAdAdTdGdGdAdCdCdAGb ⁶ Tb ⁶ Ab ⁶ Tb ⁶ Tb ⁶ 4215 19226c Tb ¹ sC*b ¹ sAb ¹ sTb ¹ sdGsdAsdAsdTsdGsdGsdAsdCsdCsdAsdGsdTsAb ¹sTb ¹ sTb ¹ 4215 19 226d Tb ¹sdCsdAsdTsdGsdAsdA*sdUsdGsdGsdAsdCsdCsdAsGb ¹ sTb ¹ sAb ¹ sTb ¹ sTb ¹4215 19 226e Tb ⁴ sC*b ⁴ sdAsdUsdGsdAsdAsdUsdGsdGsdAsdCsdC*sdAsdGsTb ⁴sAb ⁴ sTb ⁴ sTb ⁴ 4215 19 226f Tb ² ssC*b ² ssAb ² ssTb ² ssGb ²ssdAssdAssdTssdGssdGssdAssdCssdCssdAss dGssdTssdAssTb ² ssTb ² 4215 20227a C*b ¹ sTb ¹ sC*b ¹ sAb ¹ sTb ¹ sdGsdAsdAsdTsdGsdGsdAsdCsdCsdAsGb ¹sTb ¹ sAb ¹ sTb ¹ sTb ¹ 4215 20 227b C*b ² sTb ² sC*b ²sdAsdTsdGsdAsdAsdTsdGsdGsdAsdCsdCsdAsGb ² sTb ² sAb ² sTb ² sTb ² 421520 227c C*b ¹ Tb ¹ C*b ¹dAdTdGdAdAdTdGdGdAdCdC*dAdGTb ¹ Ab ¹ Tb ¹ Tb ¹4215 20 227d C*b ¹ sdUsdCsdAsdTsdGsdAsdAsdTsdGsdGsdAsdCsdCsdAsGb ¹ sTb ¹sAb ¹ sTb ¹ sTb ¹ 4215 20 227e C*b ⁴ sTb ⁴ sC*b ⁴ sAb ⁴sdTsdGsdAsdAsdTsdGsdGsdAsdCsdCsdAsdGsTb ⁴ sAb ⁴ sTb ⁴ sTb ⁴ 4214 22 228aTb ¹ sC*b ¹ sTb ¹ sC*b ¹ sAb ¹ sdTsdGsdAsdAsdTsdGsdGsdAsdCsdCsdAsdGsTb ¹sAb ¹ sTb ¹ sTb ¹ sC*b ¹ 4214 22 228b Tb ¹ C*b ¹ Tb ¹ C*b ¹ Ab¹dTdGdAdAdTdGdGdAdC*dC*dAdGTb ¹ Ab ¹ Tb ¹ Tb ¹ C*b ¹ 4214 22 228c Tb ⁶sC*b ⁶ sTb ⁶ sdCsdAsdTsdGsdAsdAsdTsdGsdGsdAsdCsdCsdAsdGsdTs Ab ⁶ sTb ⁶sTb ⁶ sC*b ⁶ 4213 24 229a Ab ¹sTb¹ sC*b ¹ sTb ¹ sC*b ¹sdAsdTsdGsdAsdAsdTsdGsdGsdAsdC*sdCsdAsdG sdTsAb ¹ sTb ¹ sTb ¹ sC*b ¹ sTb¹ 4213 24 229b Ab ¹ Tb ¹ C*b ¹ Tb ¹ C*b ¹AdTdGdAdAdTdGdGdAdCdCdAdGdTAb ¹Tb ¹ Tb ¹ C*b ¹ Tb ¹ 4212 26 230a Tb ¹ sAb ¹ sTb ¹ sC*b ¹ sTb ¹sdCsdAsdTsdGsdAsdAsdTsdGsdGsdAsdCsdCsdAs dGsdTsdAsTb ¹ sTb ¹ sC*b ¹ sTb¹ sAb ¹ 4212 26 230a Tb ¹ sAb ¹ ¹ sC*b ¹ sTb ¹sdCsdAsdTsdGsdAsdAsdTsdGsdGsdAsdCsdCsdAs dGsdTsdAsTb ¹ sTb ¹ sC*b ¹ sTb¹ sAb ¹ 4212 26 230b Tb ¹ Ab ¹ Tb ¹ C*b ¹ Tb¹dCdAdTdGdAdAdTdGdGdAdCdCdAdGdTdATb ¹ Tb ¹ C*b ¹ Tb ¹ Ab ¹ 4211 28 231aAb ¹ sTb ¹ sAb ¹ sTb ¹ sC*b¹sdTsdCsdAsdTsdGsdAsdAsdTsdGsdGsdAsdCsdCsdAsdGsdTsdAsdTsTb ¹ sC*b ¹ sTb ¹ sAb ¹ sGb ¹ 4211 28 231b Ab ¹ Tb ¹ Ab ¹Tb ¹ C*b ¹dTdCdAdTdGdAdAdTdGdGdAdCdCdAdGdTdAdTTb ¹ C*b ¹ Tb ¹ Ab ¹ Gb ¹

TABLE 9 Seq ID SP L No. Sequence, 5′-3′ 2358 10 284a C*b ¹ sAb ¹sdTsdTsdAsdAsdTsdA*sAb ¹ sAb ¹ 2358 10 284b C*b ¹ Ab ¹dTdTdA*dAdTdA*Ab ¹Ab ¹ 2357 12 285a Gb ¹ sC*b ¹ sAb ¹ sdTsdTsdA*sdA*sdTsdAsAb ¹ sAb ¹ sGb¹ 2357 12 285b Gb ¹ sC*b ¹ sAb ¹ sdTsdTsdA*sdA*sdTsdAsdA*sAb ¹ sGb ¹2357 12 285c Gb ¹ sC*b ¹ sdAsdTsdTsdA*sdA*sdUsdAsdA*sdA*sGb ¹ 2357 12285d Gb ¹ sdC*sdAsdTsdTsdAsdAsdTsdAsdAsAb ¹ sGb ¹ 2357 12 285e Gb ¹sdC*sdAsdTsdTsdAsdAsdTsdAsdA*sAb ¹ sGb ¹ 2357 12 285f Gb ¹ C*b ¹ Ab¹dTdTdA*dA*dTdAAb ¹ Ab ¹ Gb ¹ 2356 13 286a Gb ¹ sC*b ¹ sAb ¹ sTb ¹sdTsdAsdAsdTsdAsdAsAb ¹ sGb ¹ sTb ¹ 2356 13 286b Gb ¹ sC*b ¹ sAb ¹ sTb ¹sdTsdA*sdA*sdTsdAsdAsAb ¹ sGb ¹ sTb ¹ 2356 13 286c Gb ¹ sC*b ¹ sAb ¹sdUsdTsdAsdAsdTsdAsdAsdA*sGb ¹ sTb ¹ 2356 13 286d Gb ¹ sC*b ¹sdAsdTsdTsdAsdA*sdUsdAsdAsdAsdGsTb ¹ 2356 13 286e Gb ¹sdC*sdAsdTsdTsdAsdAsdTsdAsAb ¹ sAb ¹ sGb ¹ sTb ¹ 2356 13 286f Gb ¹sdC*sdAsdTsdTsdAsdAsdTsdA*sAb ¹ sAb ¹ sGb ¹ sTb ¹ 2356 13 286g Gb ¹ C*b¹ Ab ¹ Tb ¹dUdAdAdUdAdAAb ¹ Gb ¹ Tb ¹ 2356 14 287a Gb ¹ sGb ¹ sC*b ¹ sAb¹ sdTsdTsdA*sdAsdTsdAsAb ¹ sAb ¹ sGb ¹ sTb ¹ 2356 14 287b Gb ⁴ sGb ⁴sC*b ⁴ sAb ⁴ sdTsdTsdA*sdAsdUsdAsdAsdA*sGb ⁴ sTb ⁴ 2356 14 287c Gb ¹sdGsdCsdAsdUsdUsdAsdAsdTsdA*sdA*sdA*sdGsTb ¹ 2356 14 287d Gb ² sGb ²sC*b ² sdA*sdUsdTsdA*sdAsdTsdAsdA*sAb ² sGb ² sTb ² 2356 14 287e Gb ¹ Gb¹ C*b ¹ Ab ¹ sdTsdTsdAsdAsdTsdA*sdA*sAb ¹ Gb ¹ Tb ¹ 2356 14 287f Gb ¹sGb ¹ sdC*sdAsdTsdTsdAsdAsdTsdAsAb ¹ sAb ¹ sGb ¹ sTb ¹ 2356 14 287g Gb ¹sGb ¹ sdC*sdA*sdTsdTsdA*sdA*sdTsdA*sAb ¹ sAb ¹ sGb ¹ sTb ¹ 2356 14 287hGb ¹ Gb ¹dC*dAdTdTdAdAdTdAAb ¹ Ab ¹ Gb ¹ Tb ¹ 2356 14 287i Gb ⁴ ssGb ⁴ssdCssdAssdTssdTssdAssdAssdTssdAssAb ⁴ ssAb ⁴ ssGb ⁴ ssTb ⁴ 2356 14 287jGb ⁴ ssGb ⁴ ssdC*ssdAssdTssdTssdAssdAssdTssdAssAb ⁴ ssAb ⁴ ssGb ⁴ ssTb ⁴2355 15 288a Gb ¹ sGb ¹ sdC*sdAsdTsdTsdAsdAsdTsdAsdAsdAsGb ¹ sTb ¹ sGb ¹2355 15 288b Gb ¹ sGb ¹ sC*b ¹ sAb ¹ sdTsdTsdA*sdA*sdTsdAsdAsdAsdGsdTsGb¹ 2355 15 288c Gb ⁴ sGb ⁴ sC*b ⁴ sdAsdTsdTsdAsdAsdTsdAsdAsdAsdGsTb ⁴ sGb⁴ 2355 15 288d Gb ¹ sGb ¹ sC*b ¹ sAb ¹sdTdTdAdAdTdAdAsAb ¹ sGb ¹ sTb ¹sGb ¹ 2355 15 288e Gb ¹ Gb ¹ sdC*sdAsdTsdTsdAsdAsdTsdAsdAsAb ¹ Gb ¹ Tb ¹Gb ¹ 2355 15 288f Gb ¹ ssGb ¹ssdCssdAssdUssdUssdAssdAssdUssdAssdAssdAssdGssTb ¹ ssGb ¹ 2355 15 288gGb ¹ ssGb ¹ ssdCssdAssdTssdTssdAssdAssdTssdAssdAssdAssdGssdTssGb ¹ 235515 288h Gb ⁶ Gb ⁶ C*b ⁶dA*dTdTdAdAdUdA*dA*dAGb ⁶ Tb ⁶ Gb ⁶ 2355 15 288iGb ¹ Gb ¹ C*b ¹dAdTdTdAdAdUdAdAdAGb ¹ Tb ¹ Gb ¹ 2355 16 289a Ab ¹ sGb ¹sGb ¹ sC*b ¹ sAb ¹ sdTsdTsdA*sdA*sdTsdA*sAb ¹ sAb ¹ sGb ¹ sTb ¹ sGb ¹2355 16 289b Ab ¹ sGb ¹ sGb ¹ sdC*sdAsdTsdTsdAsdAsdTsdAsAb ¹ sAb ¹ sGb ¹sTb ¹ sGb ¹ 2355 16 289c Ab ¹ sGb ¹ sGb ¹sdC*sdA*sdTsdTsdA*sdA*sdTsdA*sAb ¹ sAb ¹ sGb ¹ sTb ¹ sGb ¹ 2355 16 289dAb ² sGb ² sGb ² sdC*sdAsdTsdTsdAsdAsdTsdAsAb ² sAb ² sGb ² sTb ² sGb ²2355 16 289e Ab ¹ sdGsdGsdC*sdAsdTsdTsdAsdAsdTsdAsAb ¹ sAb ¹ sGb ¹ sTb ¹sGb ¹ 2355 16 289f Ab ¹ sdGsdGsdC*sdAsdTsdUsdAsdAsdUsdAsAb ¹ sAb ¹ sGb ¹sTb ¹ sGb ¹ 2355 16 289g Ab ¹ sGb ¹ sGb ¹sdC*sdAsdTsdTsdAsdAsdTsdAsdAsAb ¹ sGb ¹ sTb ¹ sGb ¹ 2355 16 289h Ab ¹sGb ¹ sGb ¹ sC*b ¹ sdA*sdTsdTsdA*sdA*sdTsdA*sdAsdAsGb ¹ sTb ¹ sGb ¹ 235516 289i Ab ⁶ sGb ⁶ sGb ⁶ sdC*sdA*sdUsdTsdAsdAsdTsdAsdAsdAsGb ⁶ sTb ⁶ sGb⁶ 2355 16 289j Ab ¹ sGb ¹ sGb ¹ sdC*sdAsdTsdTsdAsdAsdTsdAsdAsdAsGb ¹ sTb¹ sGb ¹ 2355 16 289k Ab ¹ sdGsdGsdC*sdAsdTsdTsdAsdAsdTsdAsdAsdAsGb ¹ sTb¹ sGb ¹ 2355 16 289m Ab ¹ Gb ¹ Gb ¹ C*b ¹ Ab ¹dUdTdA*dA*dTdAdAdAdGTb ¹Gb ¹ 2355 16 289n Ab ¹ Gb ¹dGdC*dAdTdTdAdAdTdAdAAb ¹ Gb ¹ Tb ¹ Gb ¹ 235516 289o Ab ⁴ Gb ⁴ Gb ⁴dCdA*dTdTdAdAdTdAdA*Ab ⁴ Gb ⁴ Tb ⁴ Gb ⁴ 2355 16289p Ab ¹ ssGb ¹ ssGb ¹ ssC*b ¹ ssAb ¹ssdTssdTssdAssdAssdTssdAssdAssdAss Gb ¹ ssTb ¹ ssGb ¹ 2355 16 289q Ab ⁷sGb ⁷ sGb ⁷ sC*b ⁷ sdA*dTdTdAdAdTdAdA*sAb ⁷ sGb ⁷ sTb ⁷ sGb ⁷ 2355 17213j C*b ¹ sAb ¹ sGb ¹ sdGsdC*sdAsdTsdTsdAsdAsdTsdAsdAsdAsGb ¹ sTb ¹ sGb¹ 2355 17 213k C*b ¹ sAb ¹ sGb ¹ sdGsdCsdAsdTsdTsdAsdAsdTsdAsdAsdAsGb ¹sTb ¹ sGb ¹ 2355 17 213m C*b ¹ sAb ¹ sGb ¹sdGsdC*sdAsdTsdTsdAsdAsdTsdAsdA*sdA*sGb ¹ sTb ¹ sGb ¹ 2355 17 213n C*b ¹Ab ¹ Gb ¹dGdC*dAdTdTdAdAdTdAdAdAGb ¹ Tb ¹ Gb ¹ 2355 17 213o /5SpC3s/C*b¹ sAb ¹ sGb ¹ sdGsdC*sdAsdTsdTsdAsdAsdTsdAsdAsdAsGb ¹ sTb ¹ sGb ¹ 235517 213p C*b ¹ sAb ¹ sGb ¹ sdGsdC*sdAsdTsdTsdAsdAsdTsdAsdAsdAsGb ¹ sTb ¹sGb ¹ /3SpC3s/ 2355 17 213q /5SpC3s/C*b ¹ sAb ¹ sGb ¹sdGsdC*sdAsdTsdTsdAsdAsdTsdAsdAsdAsGb ¹ sTb ¹ sGb ¹ /3SpC3s/ 2355 17213r C*b ¹ sAb ¹ sGb ¹ sdGsdC*sdAsdTsdTsdAsdAsdUsdAsdAsdA*sGb ¹ sTb ¹sGb ¹ 2355 17 213s C*b ⁶ sAb ⁶ sGb ⁶ sdGdC*dAdTdTdAdAdTdAdAdAsGb ⁶ sTb ⁶sGb ⁶ 2355 17 213t C*b ¹ sAb ¹ sGb ¹ sdGdC*dAdTdTdAdAdTdAdAdAsGb ¹ sTb ¹sGb ¹ 2355 17 213u C*b ¹ Ab ¹ Gb ¹ sdGsdC*sdAsdUsdUsdAsdAsdUsdAsdAsdAsGb¹ Tb ¹ Gb ¹ 2355 17 213v C*b ¹ Ab ¹ Gb ¹sdGsdC*sdAsdTsdTsdAsdA*sdTsdAsdAsdA*sGb ¹ Tb ¹ Gb ¹ 2355 17 213w C*b ¹Ab ¹ Gb ¹ sdGsdC*sdAsdTsdTsdAsdAsdTsdAsdAsdAsGb ¹ Tb ¹ Gb ¹ 2355 17 213xC*b ⁷ sAb ⁷ sGb ⁷ sGb ⁷ sdC*sdAsdTsdTsdAsdAsdTsdAsdAsdAsGb ⁷ sTb ⁷ sGb ⁷2355 17 213y C*b ⁶ sAb ⁶ sGb ⁶ sGb ⁶ sdCsdAsdTsdTsdAsdAsdTsdAsdAsdAsGb ⁶sTb ⁶ sGb ⁶ 2355 17 213z C*b ⁷ sAb ⁷ sGb ⁷sdGsdCsdA*sdUsdTsdAsdAsdTsdAsdAsAb ⁷ sGb ⁷ sTb ⁷ sGb ⁷ 2355 17 213aa C*b⁴ sAb ⁴ sGb ⁴ sGb ⁴ sdC*sdA*sdTsdTsdAsdAsdTsdAsdA*sdAsdGsTb ⁴ sGb ⁴ 235517 213ab C*b ¹ sAb ¹ sGb ¹ sGb ¹ sC*b ¹sdA*sdTsdTsdA*sdA*sdTsdAsdA*sdA*sGb ¹ sTb ¹ sGb ¹ 2355 17 213ac C*b ¹sAb ¹ sGb ¹ sdGsdCsdAsdTsdTsdA*sdA*sdTsdAsAb ¹ sAb ¹ sGb ¹ sTb ¹ sGb ¹2355 17 213ad C*b ¹ sAb ¹ sdGsdGsdC*sdAsdTsdTsdAsdAsdUsdAsdAsAb ¹ sGb ¹sTb ¹ sGb ¹ 2355 17 213ae C*b ¹ ssAb ¹ ssGb ¹ssdGssdC*ssdAssdTssdTssdAssdAssdTssdAssdAssAb ¹ ssGb ¹ ssTb ¹ ssGb ¹2355 17 213af C*b ⁴ ssAb ⁴ ssGb ⁴ssdGssdCssdAssdTssdTssdA*ssdAssdTssdAssdAssdAss dGssTb ⁴ ssGb ⁴ 2355 17213ag C*b ² ssAb ² ssGb ² ssGb ²ssdCssdAssdTssdTssdAssdAssdTssdAssdAssdAss dGssdTssGb ² 2355 17 213ahC*b ¹ Ab ¹ Gb ¹ Gb ¹dCdAdTdTdAdAdUdAdAAb ¹ Gb ¹ Tb ¹ Gb ¹ 2355 17 213aiC*b ⁴ Ab ⁴ Gb ⁴ Gb ⁴dCdAdTdTdAdAdTdAdAdAdGTb ⁴ Gb ⁴ 2355 17 213aj C*b ¹Ab ¹ Gb ¹dGdCdAdTdTdAdAdUdAdAdAGb ¹ Tb ¹ Gb ¹ 2355 17 213ak C*b ¹ sAb ¹sGb ¹ sGb ¹sdCsdAsdTsdTsdAsdAsdTsdAsdAsAb ¹ sGb ¹ sTb ¹ sGb ¹ 2354 18290a C*b ¹ sAb ¹ sGb ¹ sGb ¹ sC*b ¹ sdAsdTsdTsdAsdAsdTsdA*sdAsAb ¹ sGb ¹sTb ¹ sGb ¹ sC*b ¹ 2354 18 290b C*b ¹ sAb ¹ sGb ¹ sGb ¹sdC*sdAsdTsdTsdAsdAsdTsdAsdAsdAsGb ¹ sTb ¹ sGb ¹ sC*b ¹ 2354 18 290c C*b¹ sAb ¹ sGb ¹ sGb ¹ sdC*sdAsdTsdTsdAsdAsdTsdAsdAsdAsdGsTb ¹ sGb ¹ sC*b ¹2354 18 290d C*b ¹ sAb ¹ sGb ¹ sdGsdC*sdAsdTsdTsdAsdAsdTsdAsdAsdAsGb ¹sTb ¹ sGb ¹ sC*b ¹ 2354 18 290e C*b ⁷ sAb ⁷ sGb ⁷ sGb ⁷ sC*b ⁷sdA*sdTsdTsdAsdAsdTsdAsdAsdAsdGsdTsdG sC*b ⁷ 2354 18 290f C*b ⁴ Ab ⁴ Gb⁴ Gb ⁴ sdCsdAsdTsdTsdAsdAsdTsdA*sdAsAb ⁴ Gb ⁴ Tb ⁴ Gb ⁴ C*b ⁴ 2354 18290g C*b ¹ ssAb ¹ ssGb ¹ssdGssdC*ssdAssdTssdTssdA*ssdAssdTssdA*ssdAssdA* ssdGssTb ¹ ssGb ¹ ssC*b¹ 2354 18 290h C*b ² Ab ² Gb ²dGdC*dAdTdTdAdAdTdAdAAb ² Gb ² Tb ² Gb ²C*b ² 2354 18 290i C*b ¹ Ab ¹dGdGdC*dA*dUdUdAdAdUdA*dA*Ab ¹ Gb ¹ Tb ¹ Gb¹ C*b ¹ 2354 19 291a Ab ¹ sC*b ¹ sAb ¹ sGb ¹ sGb ¹sdC*sdAsdTsdTsdAsdAsdTsdAsdAsAb ¹ sGb ¹ sTb ¹ sGb ¹ sC*b ¹ 2354 19 291bAb ¹ sC*b ¹ sAb ¹ sGb ¹ sdGsdC*sdAsdTsdTsdAsdAsdTsdAsdAsdAsdGsTb ¹ sGb ¹sC*b ¹ 2354 19 291c Ab ⁴ sC*b ⁴sdAsdGsdGsdC*sdAsdTsdTsdAsdAsdUsdAsdAsdAsGb ⁴ sTb ⁴ sGb ⁴ sC*b ⁴ 2354 19291d Ab ¹ sdC*sdAsdGsdGsdC*sdA*sdTsdTsdAsdAsdTsdAsdAsAb ¹ sGb ¹ sTb ¹sGb ¹ sC*b ¹ 2354 19 291e Ab ² ssC*b ² ssAb ² ssGb ² ssGb ²ssdCssdAssdTssdTssdAssdAssdTssdAssdAss dAssdGssdTssGb ² ssC*b ² 2354 19291f Ab ⁶ C*b ⁶ Ab ⁶ Gb ⁶ Gb ⁶dC*dAdTdTdAdAdTdAdAAb ⁶ Gb ⁶ Tb ⁶ Gb ⁶ C*b⁶ 2353 20 292a Ab ¹ sC*b ¹ sAb ¹ sGb ¹ sGb ¹sdC*sdAsdTsdTsdAsdAsdTsdAsdAsdAsGb ¹ sTb ¹ sGb ¹ sC*b ¹ sAb ¹ 2353 20292b Ab ² sC*b ² sAb ² sdGsdGsdC*sdAsdTsdTsdAsdAsdTsdAsdAsdAsGb ² sTb ²sGb ² sC*b ² sAb ² 2353 20 292c Ab ¹sdC*sdAsdGsdGsdC*sdAsdUsdUsdAsdAsdUsdAsdAsdAsGb ¹ sTb ¹ sGb ¹ sC*b ¹ sAb¹ 2353 20 292d Ab ⁴ sC*b ⁴ sAb ⁴ sGb ⁴sdGsdCsdAsdTsdTsdAsdAsdTsdAsdAsdAsdGsTb ⁴ sGb ⁴ sC*b ⁴ sAb ⁴ 2353 20292e Ab ¹ C*b ¹ Ab ¹dGdGdC*dAdTdTdAdAdTdAdAdAdGTb ¹ Gb ¹ C*b ¹ Ab ¹ 235222 293a Tb ¹ sAb ¹ sC*b ¹ sAb ¹ sGb ¹sdGsdC*sdAsdTsdTsdAsdAsdTsdAsdAsdAsdGsTb ¹ sGb ¹ sC*b ¹ sAb ¹ sAb ¹ 235222 293b Tb ¹ Ab ¹ C*b ¹ Ab ¹ Gb ¹dGdC*dAdTdTdAdAdTdAdAdAdGTb ¹ Gb ¹ C*b¹ Ab ¹ Ab ¹ 2352 22 293c Tb ⁶ sAb ⁶ sC*b ⁶sdAsdGsdGsdCsdAsdTsdTsdAsdAsdTsdAsdAsdAsdGsTb ⁶ sGb ⁶ sC*b ⁶ sAb ⁶ sAb ⁶2351 24 294a Ab ¹ sTb ¹ sAb ¹ sC*b ¹ sAb ¹sdGsdGsdC*sdAsdTsdTsdAsdAsdTsdAsdAsdAsdG sdTsGb ¹ sC*b ¹ sAb ¹ sAb ¹ sAb¹ 2351 24 294b Ab ¹ Tb ¹ Ab ¹ C*b ¹ Ab ¹dGdGdC*dAdTdTdAdAdTdAdAdAdGdTGb¹ C*b ¹ Ab ¹ Ab ¹ Ab ¹ 2350 26 295a Tb ¹ sAb ¹ sTb ¹ sAb ¹sC*b¹sdAsdGsdGsdC*sdAsdTsdTsdAsdAsdTsdAsdAsdAs dGsdTsdGsC*b ¹ sAb ¹ sAb¹ sAb ¹ sTb ¹ 2350 26 295b Tb ¹ Ab ¹ Tb ¹ Ab ¹ C*b¹dAdGdGdC*dAdTdTdAdAdTdAdAdAdGdTdGC*b ¹ Ab ¹ Ab ¹ Ab ¹ Tb ¹ 2349 28 236aAb ¹ sTb ¹ sAb ¹ sTb ¹ sAb ¹ sdC*sdAsdGsdGsdC*sdAsdTsdTsdAsdAsdTsdAsdAsdAsdGsdTsdGsdC*sAb ¹ sAb ¹ sAb ¹ sTb ¹ sGb ¹ 2349 28 236b Ab ¹ Tb ¹ Ab ¹Tb ¹ Ab ¹dC*dAdGdGdCdAdTdTdAdAdTdAdAdAdGdTdGdC*Ab ¹ Ab ¹ Ab ¹ Tb ¹ Gb ¹

Pharmaceutical Compositions

The antisense-oligonucleotides of the present invention are preferablyadministered in form of their pharmaceutically active salts optionallyusing substantially nontoxic pharmaceutically acceptable carriers,excipients, adjuvants, solvents or diluents. The medications of thepresent invention are prepared in a conventional solid or liquid carrieror diluents and a conventional pharmaceutically-made adjuvant atsuitable dosage level in a known way. The preferred preparations andformulations are in administrable form which is suitable for infusion orinjection (intrathecal, intracerebroventricular, intracranial,intravenous, intraparenchymal, intratumoral, intra- or extraocular,intraperitoneal, intramuscular, subcutaneous), local administration intothe brain, inhalation, local administration into a solid tumor or oralapplication. However also other application forms are possible such asabsorption through epithelial or mucocutaneous linings (oral mucosa,rectal and vaginal epithelial linings, nasopharyngial mucosa, intestinalmucosa), rectally, transdermally, topically, intradermally,intragastrically, intracutaneously, intravaginally, intravasally,intranasally, intrabuccally, percutaneously, sublingually application,or any other means available within the pharmaceutical arts.

The administrable formulations, for example, include injectable liquidformulations, retard formulations, powders especially for inhalation,pills, tablets, film tablets, coated tablets, dispersible granules,dragees, gels, syrups, slurries, suspensions, emulsions, capsules anddeposits. Other administratable galenical formulations are also possiblelike a continuous injection through an implantable pump or a catheterinto the brain.

As used herein the term “pharmaceutically acceptable” refers to anycarrier which does not interfere with the effectiveness of thebiological activity of the antisense-oligonucleotides as activeingredient in the formulation and that is not toxic to the host to whichit is administered. Examples of suitable pharmaceutical carriers arewell known in the art and include phosphate buffered saline solutions,water, emulsions, such as oil/water emulsions, various types of wettingagents, sterile solutions etc. Such carriers can be formulated byconventional methods and the active compound can be administered to thesubject at an effective dose.

An “effective dose” refers to an amount of the antisense-oligonucleotideas active ingredient that is sufficient to affect the course and theseverity of the disease, leading to the reduction or remission of suchpathology. An “effective dose” useful for treating and/or preventingthese diseases or disorders may be determined using methods known to oneskilled in the art. Furthermore, the antisense-oligonucleotides of thepresent invention may be mixed and administered together with liposomes,complex forming agents, receptor targeted molecules, solvents,preservatives and/or diluents.

Preferred are pharmaceutical preparations in form of infusion solutionsor solid matrices for continuous release of the active ingredient,especially for continuous infusion for intrathecal administration,intracerebroventricular administration or intracranial administration ofat least one antisense-oligonucleotide of the present invention. Alsopreferred are pharmaceutical preparations in form of solutions or solidmatrices suitable for local administration into the brain. For fibroticdiseases of the lung, inhalation formulations are especially preferred.

A ready-to-use sterile solution comprises for example at least oneantisense-oligonucleotide at a concentration ranging from 1 to 10 mg/ml,preferably from 5 to 10 mg/ml and an isotonic agent selected, forexample, amongst sugars such as sucrose, lactose, mannitol or sorbitol.A suitable buffering agent, to control the solution pH to 6 to 8(preferably 7-8), may be also included. Another optional ingredient ofthe formulation can be a non-ionic surfactant, such as Tween 20 or Tween80.

A sterile lyophilized powder to be reconstituted for use comprises atleast one antisense-oligonucleotide, and optionally a bulking agent(e.g. mannitol, trehalose, sorbitol, glycine) and/or a cryoprotectent(e.g. trehalose, mannitol). The solvent for reconstitution can be waterfor injectable compounds, with or without a buffering salt to controlthe pH to 6 to 8.

Aerosol preparations suitable for inhalation may include solutions andsolids in powder form, which may be in combination with apharmaceutically acceptable carrier such as inert compressed gas, e.g.nitrogen.

A particularly preferred pharmaceutical composition is a lyophilized(freeze-dried) preparation (lyophilisate) suitable for administration byinhalation or for intravenous administration. To prepare the preferredlyophilized preparation at least one antisense-oligonucleotide of theinvention is solubilized in a 4 to 5% (w/v) mannitol solution and thesolution is then lyophilized. The mannitol solution can also be preparedin a suitable buffer solution as described above.

Further examples of suitable cryo-/lyoprotectants (otherwise referred toas bulking agents or stabilizers) include thiol-free albumin,immunoglobulins, polyalkyleneoxides (e.g. PEG, polypropylene glycols),trehalose, glucose, sucrose, sorbitol, dextran, maltose, raffinose,stachyose and other saccharides (cf. for instance WO 97/29782), whilemannitol is used preferably. These can be used in conventional amountsin conventional lyophilization techniques. Methods of lyophilization arewell known in the art of preparing pharmaceutical formulations.

For administration by inhalation the particle diameter of thelyophilized preparation is preferably between 2 to 5 pm, more preferablybetween 3 to 4 μm. The lyophilized preparation is particularly suitablefor administration using an inhalator, for example the OPTINEB® orVENTA-NEB® inhalator (NEBU-TEC, Elsenfeld, Germany). The lyophilizedproduct can be rehydrated in sterile distilled water or any othersuitable liquid for inhalation administration. Alternatively, forintravenous administration the lyophilized product can be rehydrated insterile distilled water or any other suitable liquid for intravenousadministration.

After rehydration for administration in sterile distilled water oranother suitable liquid the lyophilized preparation should have theapproximate physiological osmolality of the target tissue for therehydrated peptide preparation i.e. blood for intravenous administrationor lung tissue for inhalation administration. Thus it is preferred thatthe rehydrated formulation is substantially isotonic.

The preferred dosage concentration for either intravenous, oral, orinhalation administration is between 10 to 2000 μmol/ml, and morepreferably is between 200 to 800 μmol/ml.

For oral administration in the form of tablets or capsules, the at leastone antisense-oligonucleotide may be combined with any oral nontoxicpharmaceutically acceptable inert carrier, such as lactose, starch,sucrose, cellulose, magnesium stearate, dicalcium phosphate, calciumsulfate, talc, mannitol, ethyl alcohol (liquid forms) and the like.Moreover, when desired or needed, suitable binders, lubricants,disintegrating agents and coloring agents may also be incorporated inthe mixture. Powders and tablets may be comprised of from about 5 toabout 95 percent inventive composition.

Suitable binders include starch, gelatin, natural sugars, cornsweeteners, natural and synthetic gums such as acacia, sodium alginate,carboxymethyl-cellulose, polyethylene glycol and waxes. Among thelubricants that may be mentioned for use in these dosage forms, boricacid, sodium benzoate, sodium acetate, sodium chloride, and the like.Disintegrants include starch, methylcellulose, guar gum and the like.

Additionally, the compositions of the present invention may beformulated in sustained release form to provide the rate controlledrelease of the at least one antisense-oligonucleotide to optimize thetherapeutic effects. Suitable dosage forms for sustained release includeimplantable biodegradable matrices for sustained release containing theat least one antisense-oligonucleotide, layered tablets containinglayers of varying disintegration rates or controlled release polymericmatrices impregnated with the at least one antisense-oligonucleotide.

Liquid form preparations include solutions, suspensions and emulsions.As an example may be mentioned water or water-propylene glycol solutionsfor parenteral injections or addition of sweeteners and opacifiers fororal solutions, suspensions and emulsions.

Suitable diluents are substances that usually make up the major portionof the composition or dosage form. Suitable diluents include sugars suchas lactose, sucrose, mannitol and sorbitol, starches derived from wheat,corn rice and potato, and celluloses such as microcrystalline cellulose.The amount of diluents in the composition can range from about 5% toabout 95% by weight of the total composition, preferably from about 25%to about 75% by weight.

The term disintegrants refers to materials added to the composition tohelp it break apart (disintegrate) and release the medicaments. Suitabledisintegrants include starches, “cold water soluble” modified starchessuch as sodium carboxymethyl starch, natural and synthetic gums such aslocust bean, karaya, guar, tragacanth and agar, cellulose derivativessuch as methylcellulose and sodium carboxymethylcellulose,microcrystalline celluloses and cross-linked microcrystalline cellulosessuch as sodium croscarmellose, alginates such as alginic acid and sodiumalginate, clays such as bentonites, and effervescent mixtures. Theamount of disintegrant in the composition can range from about 1 toabout 40% by weight of the composition, preferably 2 to about 30% byweight of the composition, more preferably from about 3 to 20% by weightof the composition, and most preferably from about 5 to about 10% byweight.

Binders characterize substances that bind or “glue” powders together andmake them cohesive by forming granules, thus serving as the “adhesive”in the formulation. Binders add cohesive strength already available inthe diluents or bulking agent. Suitable binders include sugars such assucrose, starches derived from wheat, corn rice and potato; natural gumssuch as acacia, gelatin and tragacanth; derivatives of seaweed such asalginic acid, sodium alginate and ammonium calcium alginate; cellulosicmaterials such as methylcellulose and sodium carboxymethylcellulose andhydroxypropyl-methylcellulose; polyvinylpyrrolidone; and inorganics suchas magnesium aluminum silicate. The amount of binder in the compositioncan range from about 1 to 30% by weight of the composition, preferablyfrom about 2 to about 20% by weight of the composition, more preferablyfrom about 3 to about 10% by weight, even more preferably from about 3to about 6% by weight.

Lubricant refers to a substance added to the dosage form to enable thetablet, granules, etc. after it has been compressed, to release from themold or die by reducing friction or wear. Suitable lubricants includemetallic stearates, such as magnesium stearate, calcium stearate orpotassium stearate, stearic acid; high melting point waxes; and watersoluble lubricants, such as sodium chloride, sodium benzoate, sodiumacetate, sodium oleate, polyethylene glycols and D,L-leucine. Lubricantsare usually added at the very last step before compression, since theymust be present on the surfaces of the granules and in between them andthe parts of the tablet press. The amount of lubricant in thecomposition can range from about 0.05 to about 15% by weight of thecomposition, preferably 0.2 to about 5% by weight of the composition,more preferably from about 0.3 to about 3%, and most preferably fromabout 0.3 to about 1.5% by weight of the composition.

Glidents are materials that prevent caking and improve the flowcharacteristics of granulations, so that flow is smooth and uniform.Suitable glidents include silicon dioxide and talc. The amount ofglident in the composition can range from about 0.01 to 10% by weight ofthe composition, preferably 0.1% to about 7% by weight of the totalcomposition, more preferably from about 0.2 to 5% by weight, and mostpreferably from about 0.5 to about 2% by weight.

In the pharmaceutical compositions disclosed herein theantisense-oligonucleotides are incorporated preferably in the form oftheir salts and optionally together with other components which increasestability of the antisense-oligonucleotides, increase recruitment ofRNase H, increase target finding properties, enhance cellular uptake andthe like. In order to achieve these goals, theantisense-oligonucleotides may be chemically modified instead of or inaddition to the use of the further components useful for achieving thesepurposes. Thus the antisense-oligonucleotides of the invention may bechemically linked to moieties or components which enhance the activity,cellular distribution or cellular uptake etc. of theantisense-oligonucleotides. Such moieties include lipid moieties such asa cholesterol moiety, cholic acid, a thioether, hexyl-S-tritylthiol, athiocholesterol, an aliphatic chain, e.g., dodecandiol or undecylresidues, a phospholipid such as dihexadecyl-rac-glycerol ortriethylammonium-1,2-di-O-hexadecyl-rac-glycero-3H-phosphonate, apolyamine or a polyethylene glycol chain, or adamantine acetic acid, apalmityl moiety, or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety. The present invention alsoincludes antisense-oligonucleotides which are chimeric compounds.“Chimeric” antisense-oligonucleotides in the context of this invention,are antisense-oligonucleotides, which contain two or more chemicallydistinct regions, one is the oligonucleotide sequence as disclosedherein which is connected to a moiety or component for increasingcellular uptake, increasing resistance to nuclease degradation,increasing binding affinity for the target nucleic acid, increasingrecruitment of RNase H and so on. For instance, the additional region ormoiety or component of the antisense-oligonucleotide may serve as asubstrate for enzymes capable of cleaving RNA:DNA hybrids or RNA:RNAmolecules. By way of example, RNase H is a cellular endoribonucleasewhich cleaves the RNA strand of an RNA:DNA duplex. Activation of RNaseH, therefore, results in cleavage of the RNA target which is the mRNAcoding for the TGF-R_(II), thereby greatly enhancing the efficiency ofantisense-oligonucleotide inhibition of gene expression. Consequently,comparable results can often be obtained with shorter oligonucleotideswhen chimeric oligonucleotides are used.

Indications

The present invention relates to the use of theantisense-oligonucleotides disclosed herein for prophylaxis andtreatment of neurodegenerative diseases, neurotrauma, neurovascular andneuroinflammatory diseases, including postinfectious and inflammatorydisorders of the central nervous system (CNS).

The antisense-oligonucleotides of the present invention are especiallyuseful for promoting regeneration and functional reconnection of damagednerve pathways and/or for the treatment and compensation of age induceddecreases in neuronal stem cell renewal.

Thus, another aspect of the present invention relates to the use of anantisense-oligonucleotide as disclosed herein for promoting regenerationneuronal tissue by reactivating neurogenesis, allowing neuronaldifferentiation and migration, and inducing integration of new neuronsinto anatomic and functional neuronal circuits.

A further aspect of the present invention relates to the use of anantisense-oligonucleotide as disclosed herein for promoting regenerationand clinical (structural) repair in patients with damage to the nervoussystem or damage to other organ systems induced by fibrosis or loss ofstem cell turnover.

Moreover, the antisense-oligonucleotides are useful for compensation andtreatment of decreases in neuronal stem cell renewal induced by age,inflammation or a gene defect.

The antisense-oligonucleotides of the present invention inhibit theTGF-R_(II) expression and are consequently used for the treatment ofdiseases associated with up-regulated or enhanced TGF-R_(II) and/orTGF-R_(II) levels.

Thus, another aspect of the present invention relates to the use of theantisense-oligonucleotides in the prophylaxis and treatment ofneurodegenerative diseases, neuroinflammatory disorders, traumatic orposttraumatic disorders, vascular or more precisely neurovasculardisorders, hypoxic disorders, postinfectious central nervous systemdisorders, fibrotic diseases, hyperproliferative diseases, cancer,tumors, presbyakusis and presbyopie.

The term “neurodegenerative disease” or “neurological disease” or“neuroinflammatory disorder” refers to any disease, disorder, orcondition affecting the central or peripheral nervous system, includingADHD, AIDS-neurological complications, absence of the Septum Pellucidum,acquired epileptiform aphasia, acute disseminated encephalomyelitis,adrenoleukodystrophy, agenesis of the Corpus Callosum, agnosia, AicardiSyndrome, Alexander Disease, Alpers' Disease, alternating hemiplegia,Alzheimer's Disease, amyotrophic lateral sclerosis (ALS), anencephaly,aneurysm, Angelman Syndrome, angiomatosis, anoxia, aphasia, apraxia,arachnoid cysts, arachnoiditis, Arnold-Chiari Malformation,arteriovenous malformation, aspartame, Asperger Syndrome, ataxiatelangiectasia, ataxia, attention deficit-hyperactivity disorder,autism, autonomic dysfunction, back pain, Barth Syndrome, BattenDisease, Behcet's Disease, Bell's Palsy, benign essential blepharospasm,benign focal amyotrophy, benign intracranial hypertension,Bernhardt-Roth Syndrome, Binswanger's Disease, blepharospasm,Bloch-Sulzberger Syndrome, brachial plexus birth injuries, brachialplexus injuries, Bradbury-Eggleston Syndrome, brain aneurysm, braininjury, brain and spinal tumors, Brown-Sequard Syndrome, bulbospinalmuscular atrophy, Canavan Disease, Carpal Tunnel Syndrome, causalgia,cavernomas, cavernous angioma, cavernous malformation, central cervicalcord syndrome, central cord syndrome, central pain syndrome, cephalicdisorders, cerebellar degeneration, cerebellar hypoplasia, cerebralaneurysm, cerebral arteriosclerosis, cerebral atrophy, cerebralberiberi, cerebral gigantism, cerebral hypoxia, cerebral palsy,cerebro-oculo-facio-skeletal syndrome, Charcot-Marie-Tooth Disorder,Chiari Malformation, chorea, choreoacanthocytosis, chronic inflammatorydemyelinating polyneuropathy (CIDP), chronic orthostatic intolerance,chronic pain, Cockayne Syndrome Type II, Coffin Lowry Syndrome, coma,including persistent vegetative state, complex regional pain syndrome,congenital facial diplegia, congenital myasthenia, congenital myopathy,congenital vascular cavernous malformations, corticobasal degeneration,cranial arteritis, craniosynostosis, Creutzfeldt-Jakob Disease,cumulative trauma disorders, Cushing's Syndrome, cytomegalic inclusionbody disease (CIBD), cytomegalovirus infection, dancing eyes-dancingfeet syndrome, Dandy-Walker Syndrome, Dawson Disease, De Morsier'sSyndrome, Dejerine-Klumpke Palsy, dementia-multi-infarct,dementia-subcortical, dementia with Lewy Bodies, dermatomyositis,developmental dyspraxia, Devic's Syndrome, diabetic neuropathy, diffusesclerosis, Dravet's Syndrome, dysautonomia, dysgraphia, dyslexia,dysphagia, dyspraxia, dystonias, early infantile epilepticencephalopathy, Empty Sella Syndrome, encephalitis lethargica,encephalitis and meningitis, encephaloceles, encephalopathy,encephalotrigeminal angiomatosis, epilepsy, Erb's Palsy, Erb-Duchenneand Dejerine-Klumpke Palsies, Fabry's Disease, Fahr's Syndrome,fainting, familial dysautonomia, familial hemangioma, familialidiopathic basal ganglia calcification, familial spastic paralysis,febrile seizures (e.g., GEFS and GEFS plus), Fisher Syndrome, FloppyInfant Syndrome, Friedreich's Ataxia, Gaucher's Disease, Gerstmann'sSyndrome, Gerstmann-Straussler-Scheinker Disease, giant cell arteritis,giant cell inclusion disease, globoid cell leukodystrophy,glossopharyngeal neuralgia, Guillain-Barre Syndrome, HTLV-1 associatedmyelopathy, Hallervorden-Spatz Disease, head injury, headache,hemicrania continua, hemifacial spasm, hemiplegia alterans, hereditaryneuropathies, hereditary spastic paraplegia, heredopathia atacticapolyneuritiformis, Herpes Zoster Oticus, Herpes Zoster, HirayamaSyndrome, holoprosencephaly, Huntington's Disease, hydranencephaly,hydrocephalus-normal pressure, hydrocephalus (in particular TGFβ-inducedhydrocephalus), hydromyelia, hypercortisolism, hypersomnia, hypertonia,hypotonia, hypoxia, immune-mediated encephalomyelitis, inclusion bodymyositis, incontinentia pigmenti, infantile hypotonia, infantilephytanic acid storage disease, infantile refsum disease, infantilespasms, inflammatory myopathy, intestinal lipodystrophy, intracranialcysts, intracranial hypertension, Isaac's Syndrome, Joubert Syndrome,Kearns-Sayre Syndrome, Kennedy's Disease, Kinsbourne syndrome,Kleine-Levin syndrome, Klippel Feil Syndrome, Klippel-Trenaunay Syndrome(KTS), Kliver-Bucy Syndrome, Korsakoff's Amnesic Syndrome, KrabbeDisease, Kugelberg-Welander Disease, kuru, Lambert-Eaton MyasthenicSyndrome, Landau-Kleffner Syndrome, lateral femoral cutaneous nerveentrapment, lateral medullary syndrome, learning disabilities, Leigh'sDisease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome, leukodystrophy,Levine-Critchley Syndrome, Lewy Body Dementia, lissencephaly, locked-insyndrome, Lou Gehrig's Disease, lupus-neurological sequelae, LymeDisease-Neurological Complications, Machado-Joseph Disease,macrencephaly, megalencephaly, Melkersson-Rosenthal Syndrome,meningitis, Menkes Disease, meralgia paresthetica, metachromaticleukodystrophy, microcephaly, migraine, Miller Fisher Syndrome,mini-strokes, mitochondrial myopathies, Mobius Syndrome, monomelicamyotrophy, motor neuron diseases, Moyamoya Disease, mucolipidoses,mucopolysaccharidoses, multi-infarct dementia, multifocal motorneuropathy, multiple sclerosis (MS), multiple systems atrophy (MSA-C andMSA-P), multiple system atrophy with orthostatic hypotension, musculardystrophy, myasthenia-congenital, myasthenia gravis, myelinoclasticdiffuse sclerosis, myoclonic encephalopathy of infants, myoclonus,myopathy-congenital, myopathy-thyrotoxic, myopathy, myotonia congenita,myotonia, narcolepsy, neuroacanthocytosis, neurodegeneration with brainiron accumulation, neurofibromatosis, neuroleptic malignant syndrome,neurological complications of AIDS, neurological manifestations of PompeDisease, neuromyelitis optica, neuromyotonia, neuronal ceroidlipofuscinosis, neuronal migration disorders, neuropathy-hereditary,neurosarcoidosis, neurotoxicity, nevus cavernosus, Niemann-Pick Disease,O'Sullivan-McLeod Syndrome, occipital neuralgia, occult spinaldysraphism sequence, Ohtahara Syndrome, olivopontocerebellar atrophy,opsoclonus myoclonus, orthostatic hypotension, Overuse Syndrome,pain-chronic, paraneoplastic syndromes, paresthesia, Parkinson'sDisease, parmyotonia congenita, paroxysmal choreoathetosis, paroxysmalhemicrania, Parry-Romberg, Pelizaeus-Merzbacher Disease, Pena Shokeir IISyndrome, perineural cysts, periodic paralyses, peripheral neuropathy,periventricular leukomalacia, persistent vegetative state, pervasivedevelopmental disorders, phytanic acid storage disease, Pick's Disease,Piriformis Syndrome, pituitary tumors, polymyositis, Pompe Disease,porencephaly, Post-Polio Syndrome, postherpetic neuralgia,postinfectious encephalomyelitis, postural hypotension, posturalorthostatic tachycardia syndrome, postural tachycardia syndrome, primarylateral sclerosis, prion diseases, progressive hemifacial atrophy,progressive locomotor ataxia, progressive multifocalleukoencephalopathy, progressive sclerosing poliodystrophy, progressivesupranuclear palsy, pseudotumor cerebri, pyridoxine dependent andpyridoxine responsive siezure disorders, Ramsay Hunt Syndrome Type I,Ramsay Hunt Syndrome Type II, Rasmussen's Encephalitis and otherautoimmune epilepsies, reflex sympathetic dystrophy syndrome, refsumdisease-infantile, refsum disease, repetitive motion disorders,repetitive stress injuries, restless legs syndrome,retrovirus-associated myelopathy, Rett Syndrome, Reye's Syndrome,Riley-Day Syndrome, SUNCT headache, sacral nerve root cysts, Saint VitusDance, Salivary Gland Disease, Sandhoff Disease, Schilder's Disease,schizencephaly, seizure disorders, septo-optic dysplasia, severemyoclonic epilepsy of infancy (SMEI), shaken baby syndrome, shingles,Shy-Drager Syndrome, Sjogren's Syndrome, sleep apnea, sleeping sickness,Soto's Syndrome, spasticity, spina bifida, spinal cord infarction,spinal cord injury, spinal cord tumors, spinal muscular atrophy,spinocerebellar atrophy, Steele-Richardson-Olszewski Syndrome,Stiff-Person Syndrome, striatonigral degeneration, stroke, Sturge-WeberSyndrome, subacute sclerosing panencephalitis, subcorticalarteriosclerotic encephalopathy, Swallowing Disorders, Sydenham Chorea,syncope, syphilitic spinal sclerosis, syringohydromyelia, syringomyelia,systemic lupus erythematosus, Tabes Dorsalis, Tardive Dyskinesia, TarlovCysts, Tay-Sachs Disease, temporal arteritis, tethered spinal cordsyndrome, Thomsen Disease, thoracic outlet syndrome, thyrotoxicmyopathy, Tic Douloureux, Todd's Paralysis, Tourette Syndrome, transientischemic attack, transmissible spongiform encephalopathies, transversemyelitis, traumatic brain injury, tremor, trigeminal neuralgia, tropicalspastic paraparesis, tuberous sclerosis, vascular erectile tumor,vasculitis including temporal arteritis, Von Economo's Disease, VonHippel-Lindau disease (VHL), Von Recklinghausen's Disease, Wallenberg'sSyndrome, Werdnig-Hoffinan Disease, Wernicke-Korsakoff Syndrome, WestSyndrome, Whipple's Disease, Williams Syndrome, Wilson's Disease,X-Linked Spinal and Bulbar Muscular Atrophy, and Zellweger Syndrome.

Preferred examples of neurodegenerative diseases and neuroinflammatorydisorders are selected from the group comprising or consisting of:

Alzheimer's disease, Parkinson's disease, Creutzfeldt Jakob disease(CJD), new variant of Creutzfeldt Jakobs disease (nvCJD), HallervordenSpatz disease, Huntington's disease, multisystem atrophy, dementia,frontotemporal dementia, motor neuron disorders of multiple spontaneousor genetic background, amyotrophic lateral sclerosis (ALS), spinalmuscular atrophy, spinocerebellar atrophies (SCAs), schizophrenia,affective disorders, major depression, meningoencephalitis, bacterialmeningoencephalitis, viral meningoencephalitis, CNS autoimmunedisorders, multiple sclerosis (MS), acute ischemic/hypoxic lesions,stroke, CNS and spinal cord trauma, head and spinal trauma, braintraumatic injuries, arteriosclerosis, atherosclerosis, microangiopathicdementia, Binswanger' disease (Leukoaraiosis), retinal degeneration,cochlear degeneration, macular degeneration, cochlear deafness,AIDS-related dementia, retinitis pigmentosa, fragile X-associatedtremor/ataxia syndrome (FXTAS), progressive supranuclear palsy (PSP),striatonigral degeneration (SND), olivopontocerebellear degeneration(OPCD), Shy Drager syndrome (SDS), age dependant memory deficits,neurodevelopmental disorders associated with dementia, Down's Syndrome,synucleinopathies, superoxide dismutase mutations, trinucleotide repeatdisorders as Huntington's Disease, trauma, hypoxia, vascular diseases,vascular inflammations, CNS-ageing. Also age dependant decrease of stemcell renewal may be addressed.

Particularly referred examples of neurodegenerative diseases andneuroinflammatory disorders are selected from the group comprising orconsisting of:

Alzheimer's disease, Parkinson's disease, Huntington's disease,amyotrophic lateral sclerosis (ALS), hydrocephalus (in particularTGFβ-induced hydrocephalus), CNS and spinal cord trauma such as spinalcord injury, head and spinal trauma, brain traumatic injuries, retinaldegeneration, macular degeneration, cochlear deafness, AIDS-relateddementia, trinucleotide repeat disorders as Huntington's Disease, andCNS-ageing.

The antisense-oligonucleotides are also useful for prophylaxis andtreatment of fibrotic diseases. Fibrosis or fibrotic disease is theformation of excess fibrous connective tissue in an organ or tissue in areparative or reactive process. This can be a reactive, benign, orpathological state. In response to injury this is called scarring and iffibrosis arises from a single cell line this is called a fibroma.Physiologically this acts to deposit extracellular matrix, which canobliterate the architecture and function of the underlying organ ortissue. Fibrosis can be used to describe the pathological state ofexcess deposition of fibrous tissue, as well as the process ofconnective tissue deposition in healing. Fibrosis is a process involvingstimulated cells to form connective tissue, including collagen andglycosaminoglycans. Subsequently macrophages and damaged tissue betweenthe interstitium release TGF-β. TGF-(3 stimulates the proliferation andactivation of fibroblasts which deposit connective tissue. Reducing theTGF-(3 levels prevents and decreases the formation of connective tissueand thus prevents and treats fibrosis.

Examples for fibrotic diseases are

-   Lungs:    -   pulmonary fibrosis    -   idiopathic pulmonary fibrosis (idiopathic means cause is        unknown)    -   cystic fibrosis-   Liver:    -   hepatic cirrhosis of multiple origin-   Heart:    -   endomyocardial fibrosis    -   old myocardial infarction    -   atrial fibrosis-   Other:    -   mediastinal fibrosis (soft tissue of the mediastinum)    -   glaucoma (eye, ocular)    -   myelofibrosis (bone marrow)    -   retroperitoneal fibrosis (soft tissue of the retroperitoneum)    -   progressive massive fibrosis (lungs); a complication of coal        workers' pneumoconiosis    -   nephrogenic systemic fibrosis (skin)    -   Crohn's Disease (intestine)    -   keloid (skin)    -   scleroderma/systemic sclerosis (skin, lungs)    -   arthrofibrosis (knee, shoulder, other joints)    -   Peyronie's disease (penis)    -   Dupuytren's contracture (hands, fingers)    -   some forms of adhesive capsulitis (shoulder)    -   residuums after Lupus erythematodes

Thus another aspect of the present invention relates to the use of anantisense-oligonucleotide for prophylaxis and/or treatment of or to theuse of an antisense-oligonucleotide for the preparation of apharmaceutical composition for prophylaxis and/or treatment of pulmonaryfibrosis, cystic fibrosis, hepatic cirrhosis, endomyocardial fibrosis,old myocardial infarction, atrial fibrosis, mediastinal fibrosis,myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis,nephrogenic systemic fibrosis, glaucoma, such as primary open angleglaucoma, Crohn's Disease, keloid, systemic sclerosis, arthrofibrosis,Peyronie's disease, Dupuytren's contracture, and residuums after Lupuserythematodes.

Still another aspect of the present invention relates to the use of anantisense-oligonucleotide for prophylaxis and/or treatment ofhyperproliferative diseases, cancer, tumors and their metastases or tothe use of an antisense-oligonucleotide for the preparation of apharmaceutical composition for prophylaxis and/or treatment ofhyperproliferative diseases, cancer, tumors and their metastases.

Examples for hyperproliferative diseases, cancer, tumors are selectedfrom the group comprising or consisting of: adenocarcinoma, melanoma,acute leukemia, acoustic neurinoma, ampullary carcinoma, anal carcinoma,astrocytoma, basal cell carcinoma, pancreatic cancer, desmoid tumor,bladder cancer, bronchial carcinoma, non-small cell lung cancer (NSCLC),breast cancer, Burkitt's lymphoma, corpus cancer, CUP-syndrome(carcinoma of unknown primary), colorectal cancer, small intestinecancer, small intestinal tumors, ovarian cancer, endometrial carcinoma,ependymoma, epithelial cancer types, Ewing's tumors, gastrointestinaltumors, gastric cancer, gallbladder cancer, gall bladder carcinomas,uterine cancer, cervical cancer, cervix, glioblastomas, gynecologictumors, ear, nose and throat tumors, hematologic neoplasias, hairy cellleukemia, urethral cancer, skin cancer, skin testis cancer, brain tumors(gliomas, e.g. astrocytomas, oligodendrogliomas, medulloblastomas,PNET's, mixed gliomas), brain metastases, testicle cancer, hypophysistumor, carcinoids, Kaposi's sarcoma, laryngeal cancer, germ cell tumor,bone cancer, colorectal carcinoma, head and neck tumors (tumors of theear, nose and throat area), colon carcinoma, craniopharyngiomas, oralcancer (cancer in the mouth area and on lips), cancer of the centralnervous system, liver cancer, liver metastases, leukemia, eyelid tumor,lung cancer, lymph node cancer (Hodgkin's/Non-Hodgkin's), lymphomas,stomach cancer, malignant melanoma, malignant neoplasia, malignanttumors gastrointestinal tract, breast carcinoma, rectal cancer,medulloblastomas, melanoma, meningiomas, Hodgkin's disease, mycosisfungoides, nasal cancer, neurinoma, neuroblastoma, kidney cancer, renalcell carcinomas, non-Hodgkin's lymphomas, oligodendroglioma, esophagealcarcinoma, osteolytic carcinomas and osteoplastic carcinomas,osteosarcomas, ovarial carcinoma, pancreatic carcinoma, penile cancer,plasmocytoma, squamous cell carcinoma of the head and neck (SCCHN),prostate cancer, pharyngeal cancer, rectal carcinoma, retinoblastoma,vaginal cancer, thyroid carcinoma, Schneeberger disease, esophagealcancer, spinalioms, T-cell lymphoma (mycosis fungoides), thymoma, tubecarcinoma, eye/ocular tumors, urethral cancer, urologic tumors,urothelial carcinoma, vulva cancer, wart appearance, soft tissue tumors,soft tissue sarcoma, Wilm's tumor, cervical carcinoma and tongue cancer.

The term “cancer” refers preferably to a cancer selected from the groupconsisting of or comprising Lung cancer, such as Lung carcinoma, livercancer such as hepatocellular carcinoma, melanoma or malignant melanoma,pancreatic cancer, such as pancreatic epithelioid carcinoma orpancreatic adenocarcinoma, colon cancer, such as colorectaladenocarcinoma, gastric cancer or gastric carcinoma, mamma carcinoma,malignant astrocytoma, prostatic cancer, such as gastric carcinoma,leukemia, such as acute myelogenous leukemia, chronic myelogenousleukemia, monocytic leukemia, promyelocytic leukemia, lymphocyticleukemia, acute lymphoblastic leukemia, lymphocytic leukemia, and acutelymphoblastic leukemia, and lymphoma, such as histiocytic lymphoma.

For the treatment of hyperproliferative diseases, cancer, tumors andtheir metastases the antisense-oligonucleotides may be administered atregular intervals (dose intervals, DI) of between 3 days and two weeks,such as 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 days, such as about 1 week,such as 6, 7 or 8 days. Suitably at least two doses are provide with aDI period between the two dosages, such as 3, 4, 5, 6, 7, 8, 9 or 10dosages, each with a dose interval (DI) between each dose of theantisense-oligonucleotide. The DI period between each dosage may thesame, such as between 3 days and two weeks, such as 4, 5, 6, 7, 8, 9,10, 11, 12, 13 days, such as about 1 week, such as 6, 7 or 8 days.

Preferably, each dose of the antisense-oligonucleotide may be betweenabout 0.25 mg/kg-about 10 mg/kg, such as about 0.5 mg/kg, about 1 mg/kg,about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg. In some embodiments,each does of the antisense-oligonucleotide may be between about 2mg/kg-about 8 mg/kg, or about 4 to about 6 mg/kg or about 4 mg/kg toabout 5 mg/kg. In some embodiments, each does of theantisense-oligonucleotide is at least 2 mg/kg, such as 2, 3, 4, 5, 6, 7or 8 mg/kg, such as 6 mg/kg. In some embodiments the dosage regime forthe antisense-oligonucleotide may be repeated after an initial dosageregime, for example after a rest period where noantisense-oligonucleotide is administered. Such as rest period may bemore than 2 weeks in duration, such as about 3 weeks or about 4 weeks,or about 5 weeks or about 6 weeks. In some embodiments the dosageregimen for the antisense-oligonucleotide is one weekly dosage, repeatedthree, four or five times. This dosage regimen may then be repeatedafter a rest period of, for example, about 3-5 weeks, such as about 4weeks. In some embodiments, the antisense-oligonucleotide isadministered during a first dosage regimen at regular dosage intervals(DI) of between 4 and 13 days for between 2-10 administrations.

Administration of the antisense-oligonucleotide is typically performedby parenteral administration, such as subcutaneous, intramuscular,intravenous or intraperitoneal administration.

DESCRIPTION OF FIGURES

FIG. 1 shows the inhibitory effect of the antisense-oligonucleotides(ASO). The DNA is transcribed to the Pre-mRNA to which in the nucleus ofthe cell, the antisense-oligonucleotides (ASO) can bind or hybridize tothe complementary sequence within an exon (as represented by the firstASO from the right side and the first ASO from the left side) or withinan intron (as represented by the second ASO from the right side) or atallocation consisting of an area of an exon and an area of an adjacentintron (as represented by the second ASO from the left side). Bypost-transcriptional modification, i.e. the splicing, the mRNA is formedto which the ASO can bind or hybridize in the cytoplasma of the cell inorder to inhibit translation of the mRNA into the protein sequence.Thus, the ASO knock down the target gene and the protein expressionselectively.

FIG. 2 shows a nucleoside unit (without internucleotide linkage) ornucleotide unit (with internucleotide linkage) which are non-LNA unitsand which may be contained in the antisense-oligonucleotides of thepresent invention especially in the region B in case theantisense-oligonucleotide of the present invention is a gapmer.

FIG. 3 shows TGF-beta and its effects on neural stem cells, cancer stemcells, and tumors. TGFbeta inhibits neural stem cell proliferation. Itmay affect the transition to a cancer stem cell, which might escape fromTGF-beta growth control. Later in tumor progression, TGF-beta acts as anoncogene; it further promotes tumor growth by promoting angiogenesis andsuppressing the immune system. In addition, it promotes cellularmigration, thereby driving cells into metastasis.

FIG. 4 shows the antisense-oligonucleotide of Seq ID No 218b in form ofa gapmer consisting of 16 nucleotides with 3 LNA units (C*b¹ and Ab¹ andTb¹) at the 5′ terminal end and 4 LNA units (Ab¹ and Gb¹ and Tb¹ andAb¹) at the 3′ terminal end and 9 DNA nucleotides (dG, dA, dA, dT, dG,dG, dA, dC, and dC) in between the LNA segments, with phosphorothioateinternucleotiodes linkages (s) and the nucleobase 5-methylcytosine (C*)in the first LNA unit from the 5′ terminal end.

Seq ID SP L No Sequence, 5′-3′ 4217 16 218b C*b ¹ sAb ¹ sTb¹sdGsdAsdAsdTsdGsdGsdAsdC sdCsAb ¹ sGb ¹ sTb ¹ sAb ¹

FIG. 5: ASO (Seq. ID No. 218b) treatment leads to intracellular pSmad2protein reduction. Labeling with an antibody against pSmad2 (leftcolumn, red) in A549 (FIG. 5A) and ReNcell CX® (FIG. 5B) cells aftergymnotic transfer with ASO Seq. ID No. 218b for 72 h or 96 hrespectively. Nuclear DNA was stained with DAPI (central column, blue).Examination of cells was performed by fluorescence microscopy (ZeissAxio® Observer.Z1). Images were analyzed with Image J Software andCorelDRAW® X7 Software. A=untreated control, B=Ref.1, C=Seq. ID No.218b.

FIG. 6: ASO (Seq. ID No. 218c) treatment leads to intracellular pSmad2protein reduction. Labeling with an antibody against pSmad2 (leftcolumn, red) in A549 (FIG. 6A) and ReNcell CX® (FIG. 6B) cells aftergymnotic transfer with ASO Seq. ID No. 218c for 72 h or 96 hrespectively. Nuclear DNA was stained with DAPI (central column, blue).Examination of cells was performed by fluorescence microscopy (ZeissAxio® Observer.Z1). Images were analyzed with Image J Software and CorelDRAW® X7 Software. A=untreated control, B=Ref.1, D=Seq. ID No. 218c.

FIG. 7: In presence of TGF-β1, ASO (Seq. ID No. 218b) treatment leads todownregulation of TGF-R_(II) mRNA. Potent downregulation of TGF-R_(II)mRNA after gymnotic transfer of TGF-R_(II) specific ASO in TGF-β1pre-incubated (48 h) A549 (FIG. 7A) and ReNcell CX® (FIG. 7B) cells.ASOs were incubated for 72 h or 96 h in presence of TGF-β1,respectively. mRNA expression levels were quantified relative tohousekeeping gene GNB2L1 using quantitative real-time RT-PCR andnormalized to untreated controls. A=untreated control, B=Ref.1, C=Seq.ID No. 218b, E=TGF-β1, +=SEM, *p<0.05, **p<0.01 in reference to A,++p<0.01 in reference to E+B. Statistics was calculated using theOrdinary-one-way-ANOVA followed by “Tukey's” post hoc comparisons.

FIG. 8: In presence of TGF-β1, ASO (Seq. ID No. 218c) treatment leads todownregulation of TGF-R_(II) mRNA. Potent downregulation of TGF-R_(II)mRNA after gymnotic transfer of TGF-R_(II) specific ASO in TGF-β1pre-incubated (48 h) A549 (FIG. 8A) and ReNcell CX® (FIG. 8B) cells.ASOs were incubated for 72 h or 96 h in presence of TGF-β1,respectively. mRNA expression levels were quantified relative tohousekeeping gene GNB2L1 using quantitative real-time RT-PCR andnormalized to untreated controls. A=untreated control, B=Ref.1, D=Seq.ID No. 218c, E=TGF-β1, ±=SEM, *p<0.05, **p<0.01 in reference to A,++p<0.01 in reference to E+B. Statistics was calculated using theOrdinary-one-way-ANOVA followed by “Tukey's” post hoc comparisons.

FIG. 9 shows the antisense-oligonucleotide of Seq ID No 209y in form ofa gapmer consisting of 16 nucleotides with 2 LNA units (Gb¹ and Tb¹) atthe 5′ terminal end and 3 LNA units (Ab¹ and Gb¹ and C*b¹) at the 3′terminal end and 11 DNA nucleotides (dA, dG, dT, dG, dT, dT, dT, dA, dG,dG, and dG) in between the LNA segments, with phosphorothioateinternucleotiodes linkages (s) and the nucleobase 5-methylcytosine (C*)in the last LNA unit from the 5′ terminal end.

Seq ID SP L No Sequence, 5′-3′ 2064 16 209y Gb ¹ sTb ¹sdAsdGsdTsdGsdTsdTsdTsdAsdGsd GsdGsAb ¹ sGb ¹ sC*b ¹

FIG. 10 shows the antisense-oligonucleotide of Seq ID No 210q in form ofa gapmer consisting of 16 nucleotides with 4 LNA units (Gb¹ and C*b¹ andTb¹ and Ab¹) at the 5′ terminal end and 3 LNA units (Gb¹ and Tb¹ andTb¹) at the 3′ terminal end and 9 DNA nucleotides (dT, dT, dT, dG, dG,dT, dA, dG, and dTs) in between the LNA segments, with phosphorothioateinternucleotiodes linkages (s) and the nucleobase 5-methylcytosine (C*)in the second LNA unit from the 5′ terminal end.

Seq ID SP L No Sequence, 5′-3′ 2072 16 210q Gb ¹ sC*b ¹ sTb ¹ sAb¹sdTsdTsdTsdGsdGsdTsd AsdGsdTsGb ¹ sTb ¹ sTb ¹

FIG. 11: In presence of TGF-β1, ASO (Seq. ID No. 218b) treatment leadsto downregulation of CTGF mRNA. Potent downregulation of CTGF mRNA aftergymnotic transfer of TGF-R_(II) specific ASO in TGF-β1 pre-incubated (48h) A549 (FIG. 11A) and ReNcell CX® (FIG. 11B) cells. ASOs were incubatedfor 72 h or 96 h in presence of TGF-β1, respectively. mRNA expressionlevels were quantified relative to housekeeping gene GNB2L1 usingquantitative real-time RT-PCR and normalized to untreated controls.A=untreated control, B=Ref.1, C=Seq. ID No. 218b, E=TGF-β1, ±=SEM,*p<0.05, **p<0.01 in reference to A, ++p<0.01 in reference to E+B.Statistics was calculated using the Ordinary-one-way-ANOVA followed by“Tukey's” post hoc comparisons.

FIG. 12: In presence of TGF-β1, ASO (Seq. ID No. 218b) treatment leadsto reduction of CTGF cellular protein. CTGF protein expression wasreduced after gymnotic transfer of TGF-R_(II) specific ASO in TGF-β1pre-incubated (48 h) A549 (FIG. 12A) and ReNcell CX® (FIG. 12B) cells.ASOs were incubated for 72 h or 96 h in presence of TGF-β1,respectively. Cells were labeled with an antibody against CTGF (leftcolumn, red). Nuclear DNA was stained with DAPI (central column, blue).Examination of cells was performed by fluorescence microscopy (ZeissAxio® Observer.Z1). Images were analyzed with Image J Software andCorelDRAW® X7 Software. A=untreated control, B=Ref.1, C=Seq. ID. 218b,E=TGF-β1.

FIG. 13: In presence of TGF-β1, ASO (Seq. ID No. 218b) treatment leadsto intracellular pSmad2 protein reduction. pSmad2 protein expression wasreduced after gymnotic transfer of TGF-R_(II) specific ASO in TGF-β1pre-incubated (48 h) A549 (FIG. 13A) and ReNcell CX® (FIG. 13B) cells.ASOs were incubated for 72 h or 96 h in presence of TGF-β1,respectively. Cells were labeled with an antibody against pSmad2 (leftcolumn, red). Nuclear DNA was stained with DAPI (central column, blue).Examination of cells was performed by fluorescence microscopy (ZeissAxio® Observer.Z1). Images were analyzed with Image J Software andCorelDRAW® X7 Software. A=untreated control, B=Ref.1, C=Seq. ID. 218b,E=TGF-β1.

FIG. 14: In presence of TGF-β1, ASO (Seq. ID No. 218c) treatment leadsto downregulation of CTGF mRNA. Potent downregulation of CTGF mRNA aftergymnotic transfer of TGF-R_(II) specific ASO in TGF-β1 pre-incubated (48h) A549 (FIG. 14A) and ReNcell CX® (FIG. 14B) cells. ASOs were incubatedfor 72 h or 96 h in presence of TGF-β1, respectively. mRNA expressionlevels were quantified relative to housekeeping gene GNB2L1 usingquantitative real-time RT-PCR and normalized to untreated controls.A=untreated control, B=Ref.1, D=Seq. ID No. 218c, E=TGF-β1, ±=SEM,*p<0.05, **p<0.01 in reference to A, Statistics were calculated usingthe Ordinary-one-way-ANOVA followed by “Dunnett's” post hoc comparisons.Note different scales.

FIG. 15: In presence of TGF-β1, ASO (Seq. ID No. 218c) treatment leadsto reduction of CTGF cellular protein. CTGF protein expression wasreduced after gymnotic transfer of TGF-R_(II) specific ASO in TGF-β1pre-incubated (48 h) A549 cells. ASOs were incubated for 72 h inpresence of TGF-β1. Cells were labeled with an antibody against CTGF(left column, red). Nuclear DNA was stained with DAPI (central column,blue). Examination of cells was performed by fluorescence microscopy(Zeiss Axio® Observer.Z1). Images were analyzed with Image J Softwareand CorelDRAW® X7 Software. A=untreated control, B=Ref.1, D=Seq. ID.218c, E=TGF-β1.

FIG. 16: In presence of TGF-β1, ASO (Seq. ID No. 218c) treatment leadsto intracellular pSmad2 protein reduction. pSmad2 protein expression wasreduced after gymnotic transfer of TGF-R_(II) specific ASO in TGF-β1pre-incubated (48 h) A549 (FIG. 16A) and ReNcell CX® (FIG. 16B) cells.ASOs were incubated for 72 h or 96 h in presence of TGF-β1,respectively. Cells were labeled with an antibody against pSmad2 (leftcolumn, red). Nuclear DNA was stained with DAPI (central column, blue).Examination of cells was performed by fluorescence microscopy (ZeissAxio® Observer.Z1). Images were analyzed with Image J Software andCorelDRAW® X7 Software. A=untreated control, B=Ref.1, D=Seq. ID. 218c,E=TGF-β1.

FIG. 17: ASO (Seq. ID No. 218b) pretreatment and subsequent TGF-β1co-exposure leads to reduction of TGF-R_(II) membrane protein.TGF-R_(II) protein was reduced after gymnotic transfer of TGF-R_(II)specific ASO followed by co-exposure of TGF-β1 (48 h) A549 (FIG. 17A)and ReNcell CX® (FIG. 17B) cells. ASOs were incubated for 72 h or 96 h,respectively, in advance to 48 h TGF-β1 co-exposure. Cells were labeledwith an antibody against TGF-R_(II) (left column, red). Nuclear DNA wasstained with DAPI (central column, blue). Examination of cells wasperformed by fluorescence microscopy (Zeiss Axio® Observer.Z1). Imageswere analyzed with Image J Software and CorelDRAW® X7 Software.A=untreated control, B=Ref.1, C=Seq. ID. 218b, E=TGF-β1.

FIG. 18: ASO (Seq. ID No. 218b) pretreatment and subsequent TGF-β1co-exposure leads to intracellular pSmad3 protein reduction. pSmad3protein expression was reduced after gymnotic transfer of TGF-R_(II)specific ASO followed by co-exposure of TGF-β1 (48 h) A549 (FIG. 18A)and ReNcell CX® (FIG. 18B) cells. ASOs were incubated for 72 h or 96 h,respectively, in advance to 48 h TGF-β1 co-exposure. Cells were labeledwith an antibody against pSmad3 (left column, red). Nuclear DNA wasstained with DAPI (central column, blue). Examination of cells wasperformed by fluorescence microscopy (Zeiss Axio® Observer.Z1). Imageswere analyzed with Image J Software and CorelDRAW® X7 Software.A=untreated control, B=Ref.1, C=Seq. ID. 218b, E=TGF β1.

FIG. 19: ASO (Seq. ID No. 218b) enhances and TGF-β11 reducesneurogenesis in human neural precursor ReNcell CX® cells. Neurogenesismarker DCX mRNA is upregulated in ReNcell CX® cells after repeatedgymnotic transfer (2×96 h) of inventive ASOs. A strong reduction of DCXmRNA expression was recognized after an 8-day TGF-β1 exposure. mRNAlevels were quantified relative to housekeeping gene GNB2L1 usingquantitative real-time RT-PCR and normalized to untreated controls.Statistics was calculated using the Ordinary-one-way-ANOVA followed by“Tukey's” multiple post-hoc comparison. A=untreated control, B=Ref.1,C=Seq. ID No. 218b, E=TGF-β1, +=SEM, +p<0.05 in reference to C 2.5 μM,#p<0.05 in reference to C 10 μM.

FIG. 20: ASO (Seq. ID No. 218b) enhances and TGF-β11 reducesproliferation in human neural precursor ReNcell CX® cells. Proliferationmarker Ki67 protein expression is increased in ReNcell CX® cells afterrepeated gymnotic transfer (2×96 h) of inventive ASOs. Reduced Ki67protein expression was recognized after an 8-day TGF-β1 exposure. Cellswere labeled with an antibody against Ki67 (left column, green). NuclearDNA was stained with DAPI (central column, blue). Examination of cellswas performed by fluorescence microscopy (Zeiss Axio® Observer.Z1).Images were analyzed with Image J Software and CorelDRAW® X7 Software.A=untreated control, B=Ref.1, C=Seq. ID No. 218b, E=TGF-β1.

FIG. 21: Despite proliferative conditions ASO (Seq. ID No. 218b)enhances differentiation in human neural precursor ReNcell CX® cells.Neural markers NeuN (FIG. 23 A, left column, red) and 111-Tubulin (FIG.23 B, left column, red) in ReNcell CX® were observed. ASO treatment wasapplied for initial 4 days under proliferative conditions followed byfurther 4 days under either proliferative (+EGF/FGF) or differentiatingconditions (−EGF/FGF). Nuclear DNA was stained with DAPI (centralcolumn, blue). Examination of cells was performed by fluorescencemicroscopy (Zeiss Axio® Observer.Z1). Images were analyzed with Image JSoftware and CorelDRAW® X7 Software. A=untreated control, B=Ref.1,C=Seq. ID No. 218b, E=TGF-β1, +EGF/FGF=proliferation,−EGF/FGF=differentiation.

FIG. 22: ASO-mediated (Seq. ID No. 218b) rescue from TGF-β-inducedneural stem cell proliferation arrest. Human neural precursor ReNcellCX® cells proliferation was observed with or without TGF-β1 exposure for7 days followed by ASO treatment for 8 days. Upregulation of GFAP (FIG.24A), Ki67 (FIG. 24B) and DCX (FIG. 24C) mRNA 7 days after TGF-β1pre-incubation indicates recovery of stem cell proliferation. mRNAexpression levels were quantified relative to housekeeping gene GNB2L1using quantitative real-time RT-PCR and normalized to untreated control.A=untreated control, B=Ref.1, C=Seq. ID No. 218b, E=TGF-β1, ±=SEM,*p<0.05 in reference to A, Statistics were calculated using theOrdinary-one-way-ANOVA followed by “Tukey's” post hoc multiplecomparisons.

FIG. 23: ASO reduces proliferation of human lung-cancer cells (A549).Proliferation marker Ki67 protein expression is decreased in A549 cellsafter gymnotic transfer (72 h) of inventive ASOs. Reduced Ki67 proteinexpression was recognized (left column, green). Nuclear DNA was stainedwith DAPI (central column, blue). Examination of cells was performed byfluorescence microscopy (Zeiss Axio® Observer.Z1). Images were analyzedwith Image J Software and CorelDRAW® X7 Software. A=untreated control,B=Ref.1, C=Seq. ID No. 218b, E=TGF-β1.

FIG. 24: ASO reduces proliferation of several human tumor cell-lines.HPAFII, K562, MCF-7, Panc-1, and HTZ-19 cells were exposed 4×72 h toinventive ASOs and proliferation was analyzed by light microscopy(Nikon, TS-100® F LED). A=untreated control, B=Ref.1, C=Seq. ID No.218b.

FIG. 25: ASO treatment mediates neural anti-fibrotic effects andameliorates cellular stress. ReNcell CX® cells were observed afterTGF-β1-preincubation (48 h) followed by gymnotic transfer of inventiveASO and co-exposure with TGF-β1 treatment for 96 h. Cells were labeledwith an antibody against CTGF (FIG. 29A, left column, red), FN (FIG.29B, left column, green) and of Phalloidin (actin-cytoskeleton, FIG.29C, left column, red). Nuclear DNA was stained with DAPI (centralcolumn, blue). Examination of cells was performed by fluorescencemicroscopy (Zeiss Axio® Observer.Z1). Images were analyzed with Image JSoftware and CorelDRAW® X7 Software. A=untreated control, B=Ref.1,C=Seq. ID No. 218b, E=TGF-β1.

FIG. 26: ASO treatment mediates tumor anti-fibrotic effects andameliorates cellular stress. A549 cells were observed after treatmentwith either TGF-β1 or gymnotic transfer of inventive ASO (72 h). Cellswere labeled with an antibody against FN (FIG. 30A, left column, green),Phalloidin (actin-cytoskeleton, FIG. 30B, left column, red). Nuclear DNAwas stained with DAPI (central column, blue). Examination of cells wasperformed by fluorescence microscopy (Zeiss Axio® Observer.Z1). Imageswere analyzed with Image J Software and CorelDRAW® X7 Software.A=untreated control, B=Ref.1, C=Seq. ID No. 218b, E=TGF-β1.

FIG. 27: ASO treatment mediates tumor anti-fibrotic effects. A549 humanlung cancer cells were observed after TGF-β1-preincubation (48 h)followed by gymnotic transfer of inventive ASO and co-exposure withTGF-β1 treatment for 72 h. Cells were labeled with an antibody againstCTGF (FIG. 31A, left column, red) and FN (FIG. 31B, left column, green).Nuclear DNA was stained with DAPI (central column, blue). Examination ofcells was performed by fluorescence microscopy (Zeiss Axio®Observer.Z1). Images were analyzed with Image J Software and CorelDRAW®X7 Software. A=untreated control, B=Ref.1, C=Seq. ID No. 218b, E=TGF-β1.

FIG. 28: ASO treatment mediates tumor anti-fibrotic effects. A549 humanlung cancer cells were observed after TGF-β1-preincubation (48 h)followed by gymnotic transfer of inventive ASO and co-exposure withTGF-β1 treatment for 72 h. Cells were labeled with an antibody againstCTGF (FIG. 32A, left column, red) and FN (FIG. 32B, left column, green).Nuclear DNA was stained with DAPI (central column, blue). Examination ofcells was performed by fluorescence microscopy (Zeiss Axio®Observer.Z1). Images were analyzed with Image J Software and CorelDRAW®X7 Software. A=untreated control, B=Ref.1, D=Seq. ID No. 218c, E=TGF-β1.

FIG. 29 shows the antisense-oligonucleotide of Seq ID No 209x in form ofa gapmer consisting of 16 nucleotides with 2 LNA units (Gb¹ and Tb¹) atthe 5′ terminal end and 3 LNA units (Ab¹ and Gb¹ and C*b¹) at the 3′terminal end and 11 DNA nucleotides (dA, dG, dT, dG, dT, dT, dT, dA, dG,dG, and dG) in between the LNA segments, with phosphorothioateinternucleotiodes linkages (s), the nucleobase 5-methylcytosine (C*) inthe last LNA unit from the 5′ terminal end, and with —O—P(O)(S⁻)OC₃H₆OHas terminal end groups at the 5′ terminal end and at the 3′ terminalend.

Seq ID SP L No Sequence, 5′-3′ 2064 16 209x /5SpC3s/Gb ¹ sTb ¹sdAsdGsdTsdGsdTsdTsdT sdAsdGsdGsdGsAb ¹ sGb ¹ sC*b ¹/3SpC3s/

FIG. 30 shows the antisense-oligonucleotide of Seq ID No 152h in form ofa gapmer consisting of 15 nucleotides with 4 LNA units (C*b¹ and Gb¹ andAb¹ and Tb¹) at the 5′ terminal end and 3 LNA units (Ab¹ and C*b¹ andAb¹) at the 3′ terminal end and 8 DNA nucleotides (dA, dC, dG, dC, dG,dT, dC, and dC) in between the LNA segments, with phosphorothioateinternucleotiodes linkages (s) and the nucleobase 5-methylcytosine (C*)in the first and second last LNA unit from the 5′ terminal end.

Seq ID SP L No Sequence, 5′-3′ 429 15 152h C*b ¹ sGb ¹ sAb ¹ sTb ¹sdAsdCsdGsdCsdGsdTsdC sdCsAb ¹ sC*b ¹ sAb ¹

FIG. 31 shows the antisense-oligonucleotide of Seq ID No 143h in form ofa gapmer consisting of 14 nucleotides with 2 LNA units (C*b¹ and Tb¹s)at the 5′ terminal end and 3 LNA units (C*b¹ and C*b¹ and Gb¹) at the 3′terminal end and 9 DNA nucleotides (dC, dG, dT, dC, dA, dT, dA, dG, anddA) in between the LNA segments, with phosphorothioate internucleotiodeslinkages (s) and the nucleobase 5-methylcytosine (C*) in the first,third from last and second LNA unit from the 5′ terminal end.

Seq ID SP L No Sequence, 5′-3′ 355 14 143h C*b ¹ sTb ¹sdCsdGsdTsdCsdAsdTsdAsdGsdAs C*b ¹ sC*b ¹ sGb ¹

FIG. 32 shows the antisense-oligonucleotide of Seq ID No 213k in form ofa gapmer consisting of 17 nucleotides with 3 LNA units (C*b¹ and Ab¹ andGb¹) at the 5′ terminal end and 3 LNA units (Gb¹ and Tb¹ and Gb¹) at the3′ terminal end and 11 DNA nucleotides (dG, dC, dA, dT, dT, dA, dA, dT,dA, dA, and dA) in between the LNA segments, with phosphorothioateinternucleotiodes linkages (s) and the nucleobase 5-methylcytosine (C*)in the first LNA unit from the 5′ terminal end.

Seq ID SP L No Sequence, 5′-3′ 2355 17 213k C*b ¹ sAb ¹ sGb ¹sdGsdCsdAsdTsdTsdAsdAsdT sdAsdAsdAsGb ¹ sTb ¹ sGb ¹

EXAMPLES

Material and Methods

Most Antisense-Oligonucleotides as well as control or referenceoligonucleotides used herein were synthesized by EXIQON as customoligonucleotides according to the needs of the inventors/applicant.Oligonucleotides having the following sequences were used as references:

(Seq. ID No. 147c) Ref.0 = dCsdAsdGsdCsdCsdCsdCsdCsdGsdAsdCsdCsdCsdAsdTsdG; (Seq. ID No. 76) Ref. 1= Ab1sAb1sC*b1sdAsdCsdGsdTsdCsdTsdAsdTsdAs C*b1sGb1sC*b1; (Seq. ID No.147m) Ref. 2 = C*b1sAb1sGb1sdCsdCsdCsdCsdCsdGsdAsdCsdC sdCsAb1sTb1sGb1;(Seq. ID No. 80) Ref. 3 = TTGAATATCTCATGAATGGA; having 2′-MOE-wings (5units 5′ and 3′) and phosphorothioate linkages.

Standard Procedures Protocols

Cell Culture:

TABLE 10 The following human cell lines were used for antisense-oligonucleotide experiments: Cell CO₂- Description line Content MediumMelanoma HTZ-19 5% DMEM F12 (Gibco 31331-018) + 1% dM-Mix (Transferrin(30 mg/ml in water 835 μl, non-essential AS (100x) 10 ml,Sodium-selenite (0.2 mg/ml in water) 70 μl, 10 ml PBS), 1% P/S Lungcarcinoma A549 5% Kaighn's F12 K + 10% FCS + 1% P/S hepatocellular HepG25% DMEM (Sigma D6429) + 10% FCS + 1% P/S carcinoma hepatocellular Hep3B5% DMEM (Sigma D6429) + 10% FCS + 1% P/S carcinoma pancreatic Panc-1 5%DMEM (Sigma D6429) + 10% FCS + 1% P/S epithelioid carcinoma pancreaticHPAFII 5% DMEM (Sigma D5796) + 15% FCS, 1% P/S, 1% adenocarcinomaAntibiotic/Antimycotic, 1% MEM Vitamin Solution, 1% non-essential AS(100x) pancreatic BxPC-3 5% RPMI (Gibco A10491-01) + 10% FCS + 1% P/S +1% adenocarcinoma Antibiotic/Antimycotic, 1% MEM Vitamin Solutionpancreatic cancer L3.6pl 5% DMEM (Sigma D5796) + 15% FCS, 1% P/S, 1%liver metastasis Antibiotic/Antimycotic, 1% Vitamin, 1% non-essential AS(100x) colorectal HT-29 5% DMEM (Sigma D5796) + 15% FCS, 1% P/S, 1%adenocarcinoma Antibiotic/Antimycotic, 1% MEM Vitamin Solution, 1%non-essential AS (100x) epithelial CaCo2 5% DMEM (Sigma D5796) + 20%FCS + 1% P/S colorectal adenocarcinoma gastric carcinoma TMK-1 5% DMEM(Sigma D5796) + 10% FCS + 1% P/S, 1% Antibiotic/Antimycotic, 1% MEMVitamin Solution malignant HTZ- 5% DMEM (Sigma D6046) + 10% FCS + 1%P/S + 1% astrocytoma 243 non-essential AS + 1% MEM Vitamin SolutionMamma- MCF-7 5% DMEM (Sigma D6046) + 10% FCS + 1% P/S Carcinomaprostatic PC-3M 5% RPMI (Gibco #61870-010), 10% FCS, 1% Sodiumadenocarcinoma pyruvate, 1% Sodium bicarbonate, 1% P/S acute KG-1 5%RPMI (Gibco #61870-010) + 10% FCS + 1% P/S myelogenous leukemia chronicK562 5% RPMI (Gibco #61870-010) + 10% FCS + 1% P/S myelogenous leukemiamonocytic THP-1 5% RPMI (Gibco #61870-010) + 10% FCS + 0.5% P/S leukemiapromyelocytic HL60 5% RPMI (Gibco #61870-010) + 10% FCS + 0.5% P/Sleukemia lymphocytic CEM- 5% RPMI (Gibco #61870-010) + 10% FCS + 0.5%P/S leukemia C7H2 acute Pre- 5% RPMI (Gibco #61870-010) + 10% FCS + 0.5%P/S lymphoblastic B697 leukemia histiocytic U937 5% RPMI (Gibco#61870-010) + 10% FCS + 0.5% P/S lymphoma Neuronal ReNcell 5% ReNcellNeural Stem Cell Maintenance Medium precursor cells of CX (Millipore#SCM005) + human FGF Basic human + cortical brain human EGF +N2-Supplement region

Material:

FCS (ATCC #30-2020)

Sodium pyruvate (Sigma #S8636)Sodium bicarbonate (Sigma #S8761-100ML)

Transferrin (Sigma #T8158-100MG) Natrium Selenite (Sigma #55261-10G)Penicillin/Streptomycin (P/S) (Sigma-Aldrich #P4458) Non-essential AminoAcids (AS) 100x (Sigma #M7145) Antibiotic/Antimycotic (Sigma #A5955) MEMVitamin Solution (Sigma #M6895) PBS (Sigma #D8537)

FGF Basic human (Millipore #GF003)EGF human (Millipore #GF144)

N-2 Supplement (Life Technologies #17502048) ReNcell Neural Stem CellMaintenance Medium (Millipore #SCM005)

Culturing and Disseminating Cells:

After removing the medium, cells were washed with PBS and incubated withaccutase (Sigma-Aldrich #P4458) (5 min, RT). Following incubation, cellswere peened and full medium (3 ml, company: see Tab. 10 for respectivecell lines) was added. Afterwards, cells were transferred into a 5 mlEppendorf Cup and centrifuged (5 min, 1000 rpm, RT). Pellet from 1T75-bottle (Sarstedt #833.910.302) was resuspended in 2.5 ml freshmedium. Cell number of cell suspension was determined with Luna-FL™automated cell counter (Biozym #872040) by staining with acridineorange/propidium iodide assay viability kit (Biozym #872045).Laminin-coating (Millipore #CC095) of dishes was necessary for adhesionof ReNcell CX® cells before seeding the cells for experiments in aconcentration of 2 μg/cm². Laminin-PBS solution was given in therespective amount directly to wells and flasks and was incubated for 1.5h at 37° C. For experiments cells were seeded and harvested as mentionedin method part of respective experimental chapter. After overnightincubation of cells at 37° C. and 5% CO₂, cells were treated asexplained in respective experimental description. 500 μl of remainingcell suspension was given into a new T75-bottle filled with 10 ml freshfull medium for culturing cells.

RNA-Analysis

Total RNA for cDNA synthesis was isolated using innuPREP® RNA Mini Kit(Analytik Jena #845-KS-2040250) according to manufacturer'sinstructions. In order to synthesize cDNA, total RNA content wasdetermined using a photometer (Eppendorf, BioPhotometer D30#6133000907), diluted with nuclease-free water. Afterwards first-strandcDNA was prepared with iScript™ cDNA Synthesis Kit (BioRad #170-8891)according to manufacturer's recommendations. For mRNA analysis real-timeRT-PCR was performed using a CFX96 Touch™ Real Time PCR Detection System(BioRad #185-5196).

All primer pairs were ready-to-use standardized and were mixed with therespective ready-to-use Mastermix solution (SsoAdvanced™ UniversialSYBR® Green Supermix (BioRad #172-5271) according to manufacturer'sinstructions (BioRad Prime PCR Quick Guide). Primer-pairs for in vivoexperiments were adapted according to individual species.

TABLE 11 Primer pairs used for mRNA Analysis Primer pair Company UniqueAssay ID Human CDKN1A BioRad qHsaCID0014498 Human CDNK1B BioRadqHsaCID0012509 Human CFLAR BioRad qHsaCID0038905 Human Col4A1 BioRadqHsaCID0010223 Human CTGF BioRad qHsaCED0002044 Human DCX BioRadqHsaCID0010869 Human FN1 BioRad qHsaCID0012349 Human GFAP BioRadqHsaCID0022307 Human GNB2L1 BioRad qHsaCEP0057912 Human ID-2 BioRadqHsaCED0043637 Human MKi67 BioRad qHsaCID0011882 Human Nestin BioRadqHsaCED0044457 Human SERPINE1 BioRad qHsaCED0043144 Human SOX2 BioRadqHsaCED0036871 Human TGFβ-RII BioRad qHsaCID0016240 Human TP53 BioRadqHsaCID0013658

As template, 1 μl of respective cDNA was used. RNA that was not reversetranscribed served as negative control for real-time RT-PCR. Forrelative quantification housekeeping gene Guanine nucleotide-bindingprotein subunit beta-2-like 1 (GNB2L1) was used. Real-time RT-PCR wasperformed with the following protocol:

TABLE 12 Protocol for real-time RT-PCR. Initiation period  2 min 95° C. 1x Denaturation  5 s 95° C. 40x Annealing, 30 s 60° C. 40x ExtensionMelting curve 65° C.-95° C.  1x (0.5° C. gradient)

Afterwards, BioRad CFX Manager 3.1 was used for quantification ofrespective mRNA-level relative to GNB2L1 mRNA and then normalized tountreated control.

Western Blot:

For protein analysis, cells/tissues were lysed using M-PER® MammalianProtein Extraction Reagent/T-PER® Tissue Protein Extraction Reagent(Thermo Scientific, #78501/#78510) according to manufacturerinstructions, respectively. SDS-acrylamide-gels (10%) were producedusing TGX Stain Free™ Fast Cast™ Acrylamide Kit (BioRad #161-0183)according to manufacturer instructions. Protein samples (20 μl) werediluted 1:5 with Lammli-buffer (6.5 μl, Roti®-Load1, Roth #K929.1),incubated at 60° C. for 30 min and loaded on the gel with the entirevolume of the protein solution. Separation of proteins was performed byelectrophoresis using PowerPac™ Basic Power Supply (Biorad #164-5050SP)and Mini-PROTEAN® Tetra cell electrophoresis chamber (BioRad#165-8001-SP) (200 V, 45 min). Following electrophoresis, the proteinswere blotted using Trans-Blot® Turbo Transfer System (BioRad#170-4155SP). All materials for western blotting were included inTrans-Blot® Turbo RTA PVDF-Midi Kit (BioRad #170-4273).

The PVDF-membrane for blotting procedure was activated in methanol(Merck #1.06009.2511) and equilibrated in 1× transfer buffer. Followingblotting (25 V, 1 A, 30 min), membranes were washed (3×, 10 min, RT)with 1×TBS (Roth #10.60.1) containing 0.5 ml Tween-20 (Roth #9127.1).Afterwards, the membranes were blocked with 5% BSA (Albumin-IgG-free,Roth #3737.3) diluted with TBS-T for 1 h at RT, the primary antibodies(diluted in 0.5% BSA in TBS-T, Table 13) were added and incubated at 4°C. for 2 days. Antibodies for in vivo experiments were chosen forspecies specificity accordingly.

TABLE 13 Antibodies used for Western Blot analysis. Dilution CompanyOrder Number Primary Antibody Alpha-Tubulin HRP-linked 1:2000 CellSignaling cs12351s (rabbit) ColIV (rabbit) 1:1000 Abcam ab6586 CTGF(rabbit) 1:1000 Genetex GTX-26992 FN (rabbit) 1:250 Proteintech15613-1-AP GAPDH XP HRP-linked 1:1000 Cell Signaling cs8884s (rabbit)Ki67 (rabbit) 1:500 Abcam ab15580 pAkt (rabbit) 1:1000 Cell signalingcs4060s pErk1/2 (rabbit) 1:1000 Cell signaling cs4370s pSmad2 (rabbit)1:500 Cell Signaling cs3104 TGF-βRII (rabbit) 1:400 Aviva ARP44743-T100Secondary Antibody Anti-rabbit IgG, HRP- 1:10000 Cell signalingcs#12351S linked

In the next step, membranes were washed in TBS-T (3×10 min, RT) andincubated with the secondary antibody (1h, RT, Table 13). Followingincubation, blots were washed with TBS-T, emerged using Luminata™ ForteWestern HRP Substrate (Millipore #WBLUF0500) and bands were detectedwith a luminescent image analyzer (ImageQuant™ LAS 4000, GE Healthcare).Afterwards, the blots were washed in TBS-T (3×10 min, RT) and blockedwith 5% BSA diluted in TBS-T (1h, RT). For housekeeper comparison, themembranes were incubated with HRP-conjugated anti alpha-tubulin (1:2000in 0.5% BSA, 4° C., overnight). The next day blots were emerged usingLuminata™ Forte Western HRP Substrate (Millipore #WBLUF0500) and bandswere detected with the luminescent image analyzer. Finally, the blotswere washed with TBS-T (3×, 5 min) and stained using 1× Roti®-Bluesolution (Roth #A152.2) and dried at RT.

Immunocytochemistry

Cells were treated and harvested as described before. Following fixationof cells with Roti®-Histofix 4% (Roth #P087.4) on 8-well, cell cultureslide dishes (6 min, RT) were washed three times with PBS. Afterblocking cells for 1 h at RT with Blocking Solution (Zytomed#ZUC007-100) cells were incubated with respective primary antibodieslisted in Table 14 and incubated at 4° C. overnight.

Afterwards, cell culture slides were washed three times with PBSfollowing incubation with secondary antibody (1 h, RT). Allantibody-dilutions were prepared with Antibody-Diluent (Zytomed#ZUC025-100).

TABLE 14 Antibodies used for immunocytochemistry. Dilution Company OrderNumber Primary Antibody ColIV (rabbit) 1:50 Abcam ab6586 CTGF (rabbit)1:50 Genetex GTX26992 βIII-Tubulin (rabbit) 1:100 cell signaling cs5568FN (rabbit) 1:50 Proteintech 15613-1-AP Ki67 (rabbit) 1:100 Abcamab15580 NeuN (rabbit) 1:250 Abcam Ab104225 Phalloidin Alexa Fluor 1:20Cell signaling cs8953 555 pSmad2 (rabbit) 1:50 Cell signaling cs3104spSmad3 (rabbit) 1:50 Cell signaling cs9520s TGF-R_(II) (rabbit) 1:50Millipore 06-227 Secondary Antibody Alexa Fluor 488 1:750 LifeTechnologies A21441 Cy3 goat-anti-rabbit 1:1000 Life Technologies A10520

Following incubation with secondary antibody, cells were washed threetimes with PBS, coverslips were separated from cell culture dish andmounted with VECTASHIELD® HardSet™ with DAPI (Biozol #VEC-H-1500).Slides were dried overnight at 4° C. before fluorescence microscopy(Zeiss, Axio® Observer.Z1). Images were analyzed with Image J Softwareand CorelDRAW® X7 Software.

In Vivo Experiments

Peripheral Blood Mononuclear Cell (PBMC) Assay

PBMCs were isolated from buffy coats corresponding to 500 ml full bloodtransfusion units. Each unit was obtained from healthy volunteers andglucose-citrate was used as an anti-agglutinant. The buffy coat bloodwas prepared and delivered by the Blood Bank Suhl of the Institute forTransfusion Medicine, Germany. Each blood donation was monitored for HIVantibody, HCV antibody, HBs antigen, TPHA, HIV RNA, and SPGT (ALAT).Only blood samples tested negative for infectious agents and with anormal SPGT value were used for leukocyte and erythrocyte separation bylow-speed centrifugation. The isolation of PBMCs was performed about 40h following blood donation by gradient centrifugation usingFicoll-Histopague® 1077 (Heraeus™ Multifuge™ 3 SR). For IFNα assay,PBMCs were seeded at 100,000 cells/96-well in 100 μl complete mediumplus additives (RPM11640, +L-Glu, +10% FCS, +PHA-P (5 μg/ml), +IL-3 (10μg/ml)) and test compounds (5 μl) were added for direct incubation (24h, 37° C., 5% CO₂). For TNFα assay, PBMCs were seeded at 100,000cells/96-well in 100 μl complete medium w/o additives (RPM11640, +L-Glu,+10% FCS) and test compounds (5 μl) were added for direct incubation (24h, 37° C., 5% CO₂). ELISA (duplicate measurement out of pooledsupernatants, 20 μl) for huIFNα (eBioscience, #BMS216INSTCE) wasperformed according to the manufacturer's protocol. ELISA (duplicatemeasurement out of pooled supernatants, 20 μl) for huTNFα (eBioscience,#BMS2231NSTCE) was performed according to the manufacturer's protocol.

bDNA Assay

TGF-R_(II) mRNA levels were determined in liver, kidney, and lung lysateby bDNA assay according to manufacturer's instructions (QuantiGene® kit,Panomics/Affimetrix).

Immunofluorescence

Paraffin-embedded spinal cord and brain tissue was cut into 5 μmsections (3-4 slides per object plate). Paraffin sections weredeparaffinized and demasked by heating in citrate buffer (10 mM, 40 min)in a microwave oven. Afterwards, deparaffinized sections were incubatedwith 0.3% H₂O₂ (30 min, RT), washed with PBS (10 min, RT) and blockedwith Blocking Solution (Zytomed #ZUC007-100) for 30 min. After blockingfor 1 h at RT with Blocking Solution (Zytomed) slides were incubatedwith 150 μl of the respective primary antibodies and incubated at 4° C.overnight. After washing with PBS (three times, 5 min RT) the sliceswere incubated with the secondary antibody for 1 h at RT. All antibodydilutions were prepared with Antibody Diluent (Zytomed #ZUC025-100).Afterwards the slices were washed again with PBS (three times, 5 min,RT) and mounted using VECTASHIELD® Mounting Medium with DAPI (Vector).Antibodies for immunofluorescence were comparable to cell cultureexperiments and adapted for each species.

Electrochemiluminescence

For immunological and hematological alterations,electrochemiluminescence technique (MesoScale Discovery®, Maryland,United States) was used. For each assay, 25 μl of the protein, blood,and liquor samples were used and the procedure was performed accordingto manufacturer's instructions.

BrdU Assay

Labeling of dividing cells was performed by intraperitoneal injection ofthe thymidine analogue BrdU (Sigma, Steinheim, Germany) at 50 mg/kg ofbody weight using a sterile solution of 10 mg/ml of BrdU dissolved in a0.9% (w/v) NaCl solution. The BrdU injections were performed dailywithin the last experimental week.

Surgery

For chronic central infusion, animals underwent surgery for an icycannula attached to an Alzet® osmotic minipump (mice, rats, infusionrate: 0.25 μl/h, Alzet®, Model 2004, Cupertino, USA) or a gas pressurepump (Cynomolgus monkeys, infusion rate 0.25 ml/24 h, Tricumed®, ModelIP 2000V, Germany). The cannula and the pump were stereotaxicallyimplanted under ketamine/xylacin anesthesia (Baxter, GmbH, Germany) andsemi-sterile conditions. Each osmotic minipump/gas pressure pump wasimplanted subcutaneously in the abdominal region via a skin incision atthe neck of the animals and connected with the icy cannula by siliconetubing. Animals were placed into a stereotaxic frame, and the icycannula was lowered into the right lateral ventricle. The cannula wasfixed with two stainless steel screws using dental cement (Kallocryl,Speiko®-Dr. Speier GmbH, Monster, Germany). The skin of the neck wasclosed with sutures. During surgery, the body temperature was maintainedby a heating pad. To avoid post-surgical infections, animals werelocally treated with Betaisodona® (Mundipharma GmbH, Limburg, Germany)and received antibiotics (sc, Baytril® 2.5% Bayer Vital GmbH,Leverkusen, Germany). The tubing was filled with the respectivesolution. Blood, liquor, and tissues were collected for analysis.Histological verification of the icy implantation sites was performed at40 μm coronal, cresyl violet-stained brain sections.

Outcome Parameters and Functional Analysis

Onset of symptomatic disease, onset of first paresis and survival wereused as in vivo endpoints. Onset of symptomatic disease was defined as alack of leg stretching in reaction to tail suspending. Time point atwhich gait impairments were first detected (e.g., hobbling or waddling)was classified as onset of first paresis. These parameters weredetermined daily starting at age 40 days.

To monitor disease progression, running wheel testing (LMTB, Berlin,Germany) was performed. Animals were caged separately with access to arunning wheel starting at 33 days of age. Motor activity was directlycorrelated with the rotations per minute, generated by each animal inthe running wheel. Each full turn of the wheel triggered twoelectromagnetic signals, directly fed into a computer attached to amaximum of 120 wheels. Running wheel data were recorded and analyzedwith “Maus Vital” software (Laser- und Medizin-Technologie, Berlin,Germany). Assessment time lasted for 12 hours from 6:00 pm to 6:00 am.

Spatial Learning Test (Morris-Water-Maze)

Behavioral testing was performed between 8:00 and 13:00.

Rats were trained in a black circular pool (1.4 m in diameter, 50 cmhigh, filled with 20° C. warm water to a height of 30 cm) to find avisible white target (10 cm in diameter, raised above the water'ssurface of approximately 1 cm) that was located throughout the study inthe center of the same imaginary quadrant (proximally cued). Each animalwas trained to navigate to the platform in 3 consecutive sessions with12 trials/sessions, one session per day and an inter-trial interval of10-20 s.

Microbiological Analysis

Antisense-oligonucleotide samples were microbiologically analyzedaccording to Ph. Eur. 2.6.12, USP 30<61> regarding the Total AerobicMicrobial Count (TAMC) and the Total Combined Yeast and Mould Count(TYMC).

Anion-Exchange High-Performance Liquid Chromatography (AEX-HPLC)

Integrity and stability of antisense-oligonucleotide (ASO) samples wasdetermined by AEX-HPLC using AKTAexplorer™ System (GE healthcare,Freiburg, Germany). The purified ASO samples were desalinated by ethanolprecipitation. The identity of the ASO was confirmed byelectrospray-ionization-mass-spectrometry (ESI-MS) and the purity wasdetermined by AEX-HPLC with a Dionex DNAPac™ 200 (4×250 mm) column.

Example 1: Determination of Inhibitory Activity of InventiveAntisense-Oligonucleotides on mRNA Level

1.1 Transfection of Antisense-Oligonucleotides

The inhibitory activity of several antisense-oligonucleotides directedto TGF-R_(II) was tested in human epithelial lung cancer cells (A549).TGF-R_(II) mRNA was quantified by branched DNA assay in total mRNAisolated from cells incubated with TGF-R_(II) specific oligonucleotides.

Description of Method:

Cells were obtained and cultured as described above. Transfection ofantisense-oligonucleotides was performed directly after seeding 10,000A549 cells/well on a 96-well plate, and was carried out withLipofectamine® 2000 (Invitrogen GmbH, Karlsruhe, Germany, cat. No.11668-019) as described by the manufacturer. In two independent singledose experiments performed in quadruplicates, oligonucleotides weretransfected at a concentration of 20 nM. After transfection, cells wereincubated for 24 h at 37° C. and 5% CO₂ in a humidified incubator(Heraeus GmbH, Hanau, Germany). For measurement of TGF-R_(II) mRNA,cells were harvested and lysed at 53° C. following proceduresrecommended by the manufacturer of the QuantiGene® Explore Kit(Panomics, Fremont, Calif., USA, cat. No. QG0004) for isolation ofbranched DNA (bDNA). For quantitation of housekeeping geneGlyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA the QuantiGene®Explore Kit was used, whereas quantitation of TGF-R_(II) mRNA wasconducted with QuantiGene® 2.0 (custom manufacturing for Axolabs GmbH,Kulmbach, Germany). After incubation and lysis, 10 μl of the lysateswere incubated with probe sets specific to human TGF-R_(II) and humanGAPDH. Both reaction types were processed according to themanufacturer's protocol for the respective QuantiGene® kit.Chemoluminescence was measured in a Victor²™ multilabel counter (PerkinElmer, Wiesbaden, Germany) as RLUs (relative light units) and valuesobtained with the TGF-R_(II) probe sets were normalized to therespective GAPDH values for each well and then normalized to thecorresponding mRNA readout from mock-treated cells.

Results

Results show the efficient downregulation of TGF-R_(II) by several ASOsafter transfection of A549 cells.

TABLE 15 Downregulation of TGF-R_(II) mRNA. Transfection with TGF-R_(II)specific antisense-oligonucleotides (ASOs) in human epithelial lungcarcinoma cells (A549). Quantitation of mRNA expression levels wasperformed relative to housekeeping gene GAPDH using QuantiGene ® Kit.Probes were then normalized to the corresponding mRNA readout frommock-treated cells. A549 (c = 20 nM) GAPDH TGF-R_(II) ASO mean SD meanSD Seq. ID No. 141j 1.41 0.05 0.02 0.01 Seq. ID No. 143aj 0.76 0.03 0.020.01 Seq. ID No. 139c 0.9 0.03 0.02 0.01 Seq. ID No. 145c 0.91 0.05 0.030.01 Seq. ID No. 209ax 1.52 0.58 0.03 0.01 Seq. ID No. 152ak 0.88 0.030.04 0 Seq. ID No. 218ar 1.08 0.03 0.04 0 Seq. ID No. 144c 0.5 0.07 0.050.03 Seq. ID No. 210ap 0.92 0.05 0.05 0.01 Seq. ID No. 142c 1.33 0.050.06 0.03 Seq. ID No. 213ak 1.2 0.03 0.07 0.01 Seq. ID No. 153f 1.090.07 0.08 0.03

Conclusion

TGF-R_(II) mRNA was efficiently targeted by the inventive ASOs. Thenamed ASOs achieved an effective target mRNA downregulation aftertransfection of A549 cells.

1.2 Gymnotic Uptake of Antisense-Oligonucleotides

1.2.1a Comparison of Target-Knockdown Between Inventive ASOs andPrior-Art Sequences by Gymnotic Transfer in A549 and Panc-1 Cells

The downregulatory activity of several antisense-oligonucleotidesdirected to TGF-R_(II) was tested in human epithelial lung tumor cells(A549) by direct uptake without transfection reagents (“gymnoticuptake”). TGF-R_(II) mRNA was quantified by branched DNA assay in totalmRNA isolated from cells incubated with TGF-R_(II) specificoligonucleotides.

Description of Method:

Cells were obtained and cultured as described in general methods.Gymnotic transfer of antisense-oligonucleotides was performed bypreparing a 96-well plate with the respective antisense-oligonucleotidesand subsequently seeding of 10,000 cells (Panc-1) or 8,000 cells(A549)/well. Experiments were performed in quadruplicates,oligonucleotides were used at final concentrations of 5 μM (Panc-1) and7.5 μM (A549). Cells were incubated for 72 h at 37° C. and 5% CO₀₂ in ahumidified incubator (Heraeus GmbH, Hanau, Germany). For measurement ofTGF-R_(II) mRNA, cells were harvested and lysed at 53° C. followingprocedures recommended by the manufacturer of the QuantiGene® ExploreKit (Panomics, Fremont, Calif., USA, cat. No. QG0004) for branched DNA(bDNA). For quantitation of housekeeping gene GAPDH mRNA the QuantiGene®Explore Kit was used, whereas quantitation of TGF-R_(II) mRNA wasconducted with QuantiGene® 2.0 (custom manufacturing for Axolabs GmbH,Kulmbach, Germany). After incubation and lysis, 10 μl of the lysateswere incubated with probe sets specific to human TGF-R_(II) and humanGAPDH. Both reaction types were processed according to themanufacturer's protocol for the respective QuantiGene® kit.Chemoluminescence was measured in a Victor²™ multilabel counter (PerkinElmer, Wiesbaden, Germany) as RLUs (relative light units) and valuesobtained with the TGF-R_(II) probe sets were normalized to therespective GAPDH values for each well and then normalized to thecorresponding mRNA readout from PBS treated cells.

TABLE 16a Efficacy of target mRNA downregulation by gymnotic transfer.Remaining TGF-R_(II) mRNA after gymnotic uptake of selected TGF-R_(II)specific ASOs in A549 and Panc-1 cells. mRNA expression levels weredetermined relative to housekeeping gene Glyceraldehyde- 3-phosphatedehydrogenase (GAPDH) and compared to PBS treated cells as referencecontrol (=1) using QuantiGene ® Kit. Remaining mRNA of TGF-R_(II) (PBStreated cells = 1) A549 cells Panel cells ASO mean SD mean SD Seq. IDNo. 209ay 0.11 0.01 0.07 0.02 Seq. ID No. 209ax 0.14 0.02 0.08 0.01 Seq.ID No. 209bb 0.19 0.01 0.11 0.01 Seq. ID No. 209az 0.19 0.03 0.13 0.02Seq. ID No. 209ba 0.23 0.02 0.18 0.03 Seq. ID No. 209y 0.27 0.04 0.170.01 Seq. ID No. 152h 0.29 0.04 0.12 0.02 Seq. ID No. 218b 0.30 0.020.07 0.01 Seq. ID No. 213k 0.34 0.04 0.17 0.04 Seq. ID No. 210q 0.370.05 0.18 0.02 Seq. ID No. 210aq 0.39 0.03 0.18 0.02 Seq. ID No. 143h0.43 0.04 0.35 0.05 Ref. 2 0.59 0.05 0.40 0.04 Ref. 0 0.89 0.06 1.100.07 Ref. 3 0.68 0.03 0.62 0.03

Conclusion

Gymnotic transfer of inventive ASOs results in a continuously strongerdownregulation of the target TGF-R_(II) mRNA than the transfer of testedreference sequences. The claimed antisense-oligonucleotides outperformedall tested sequences known from prior-art, independently of the chosenhuman cell line. Nevertheless, in general antisense-oligonucleotideshaving a length of 12-20 nucleotides result in a more effectivedownregulation of the target TGF-R_(II) mRNA than shorter or longerantisense-oligonucleotides. This effect was even more noticeable forantisense-oligonucleotides having a length of 14-18 nucleotides, whichin general show the most potent effects.

1.2.1b Analysis of Gymnotic Transfer in A549 Cells by Branched DNA Assay

Most effective antisense-oligonucleotides against TGF-R_(II) from thetransfection screens were further characterized by gymnotic uptake inA549 cells. TGF-R_(II) mRNA was quantified by branched DNA in total mRNAisolated from cells incubated with TGF-R_(II) specificantisense-oligonucleotides.

Description of Method:

A549 cells were cultured as described before under standard conditions.For single-dose and dose-response experiments 80,000 A549 cells/wellwere seeded in a 6-well culture dish and incubated directly witholigonucleotides at a concentration of 7.5 μM. For measurement ofTGF-R_(II) mRNA, cells were harvested, lysed at 53° C. and analyzed bybranched DNA Assay following procedures recommended by the manufacturerof the QuantiGene® Explore Kit (Panomics, Fremont, Calif., USA, cat. No.QG0004) as described above (see 1.1).

Results

Listed ASOs in Table 16b showed reduced target mRNA level of TGF-R_(II)relative to the housekeeping gene GAPDH in A549 cells. The ten mostefficient ASOs were also tested for inhibitory concentration 50 (IC₅₀).All together Seq. ID No. 209t, Seq. ID No. 218b, Seq. ID No. 218c andSeq. ID No. 209y lead to most proper knockdown of TGF-R_(II) at lowconcentration levels.

TABLE 16b Downregulation of TGF-R_(II) mRNA after gymnotic uptake ofTGF- R_(II) specific ASOs in A549 cells. mRNA levels were determinedrelative to housekeeping gene GAPDH using QuantiGene ® Kit. TGF-R_(II)GAPDH IC₅₀ ASO n = 4 SD n = 4 SD n = 4 Seq. ID No. 209t 0.19 0.05 1.130.11 1.63 Seq. ID No. 218c 0.25 0.04 0.94 0.18 1.17 Seq. ID No. 218b0.26 0.08 1.08 0.28 2.54 Seq. ID No. 218q 0.27 0.07 1.11 0.08 2.39 Seq.ID No. 209y 0.34 0.06 0.96 0.06 1.57 Seq. ID No. 218t 0.36 0.12 0.760.04 2.57 Seq. ID No. 218m 0.41 0.06 1.16 0.29 1.66 Seq. ID No. 209w0.44 0.07 1.00 0.11 5.76 Seq. ID No. 218p 0.46 0.12 0.88 0.07 Seq. IDNo. 209v 0.48 0.25 0.96 0.07 3.10 Seq. ID No. 209x 0.52 0.02 0.87 0.065.60 Seq. ID No. 218u 0.53 0.20 0.79 0.05 Seq. ID No. 218v 0.54 0.130.77 0.04 Seq. ID No. 210q 0.60 0.23 1.11 0.11 Seq. ID No. 218o 0.610.15 0.96 0.06 Seq. ID No. 210p 0.65 0.24 1.01 0.23 Seq. ID No. 218n0.89 0.36 1.07 0.22 Seq. ID No. 210o 0.95 0.08 0.97 0.14 Seq. ID No.209s 0.96 0.31 1.14 0.24 pos. Ctrl aha-1 0.22 0.04 0.77 0.02 Ref. 1 1.430.40 1.27 0.18 IC₅₀ = inhibitory concentration for 50% ofdownregulation, Pos. Ctrl: aha-1 = activator of heat shock 90 kDaprotein ATPase homolog 1 (Aha1) directed LNA as positive control, Ref. 1= Scrambled control.

Conclusion

The target downregulation by the most efficient inventive ASOs was againexcellent without transfection reagents. Thus, gymnotic transfer isfeasible and the preferred method for further drug development.

1.2.2 Analysis of Gymnotic Uptake in A549 and ReNcell CX® Cells

Inhibitory activity on the target mRNA by antisense-oligonucleotides(ASOs) was determined in human neuronal progenitor cells from corticalbrain region (ReNcell CX® cells, Millipore #SCM007). Questions regardingadult neurogenesis as therapeutic target were assessed by gymnotictransfer studies with most effective ASOs. A549 cells were used asreference cell line.

Description of Method:

A549 and ReNcell CX® cells were cultured as described above. Fortreatment studies cells were seeded in a 24-well culture dish (Sarstedt#83.1836.300) (50,000 cells/well) and were incubated overnight at 37° C.and 5% CO₂. For treatment of A549 and ReNcell CX® cells, medium wasremoved and replaced by fresh full medium (0.5 ml for 24-well). Ref.1,ASO with Seq. ID No. 218b, and ASO with Seq. ID No. 218c were then addedin medium at concentrations of 2.5 and 10 μM for analysis of targetdownregulation at different time points (A549 cells: 18 h, 72 h, 6 d,ReNcell CX® cells: 18 h, 96 h, 8 d) at 37° C. and 5% CO₂. Forharvesting, cells were washed twice with PBS and frozen at −20° C. Foranalysis of mRNA by real-time RT-PCR, cells were processed as describedabove. Ready-to-use and standardized primer pairs for real-time RT-PCR(see Table 11) were used and mixed with the respective ready-to-useMastermix solution (SsoAdvanced™ Universial SYBR® Green Supermix (BioRad#172-5271) according to manufacturer's instructions (BioRad Prime PCRQuick Guide). Probes were analyzed as triplicates and data wasquantified relative to GNB2L1 mRNA using BioRad CFX Manager™ 3.1 andthen normalized to untreated control. Statistics were calculated usingthe Ordinary-one-way-ANOVA followed by “Dunnett's” post hoc comparisons.

Results:

Results showed that gymnotic transfer with Seq. ID No. 218b and 218cresult in a proper downregulation of TGF-R_(II) mRNA in A549 and ReNcellCX® cells in a dose- and time dependent manner (Table 17). Target mRNAin A549 cells was significantly reduced after 18 h, and was even moreefficient reduced after 72 h and 6 d. After 18h in ReNcell CX® only adepression of TGF-R_(II) mRNA after gymnotic uptake of 10 μM could beobserved, but target downregulation was significant after 72 h for bothtested concentrations and was stable until day 8.

TABLE 17 Dose- and time-dependent downregulation of TGF-R_(II) mRNAafter gymnotic transfer with TGF-R_(II) specific ASO in A549 and ReNcellCX ® cells. mRNA expression levels were determined relative tohousekeeping gene GNB2L1 using quantitative real-time RT-PCR and thennormalized to untreated control. Cell line A549 Target TGF-R_(II)TGF-R_(II) TGF-R_(II) Time point 18 h, 72 h, 6 d, n = 3 n = 3 n = 3 A1.00 ± 0.03 1.00 ± 0.20 1.00 ± 0.38 B 2.5 μM 1.17 ± 0.06 0.87 ± 0.210.88 ± 0.14 B 10 μM 0.98 ± 0.10 0.77 ± 0.06 1.03 ± 0.10 C 2.5 μM 0.60*++± 0.09    0.41* ± 0.07  0.13 ± 0.03 C 10 μM 0.49**++ ± 0.02    0.15** ±0.02  0.02*+ ± 0.00   D 2.5 μM 0.46** ± 0.09  D 10 μM 0.21* ± 0.04  Cellline ReNcell CX Target TGF-R_(II) TGF-R_(II) TGF-R_(II) Time point 18 h,96 h, 8 d, n = 3 n = 3 n = 3 A 1.00 ± 0.41 1.00 ± 0.04 1.00 ± 0.18 B 2.5μM 1.38 ± 0.58 0.89 ± 0.09 0.80 ± 0.33 B 10 μM 1.70 ± 0.68 0.81 ± 0.101.16 ± 0.43 C 2.5 μM 1.04 ± 0.36 0.32** ± 0.06  0.42 ± 0.16 C 10 μM 0.64± 0.24 0.16** ± 0.02  0.21 ± 0.09 D 2.5 μM 0.53 ± 0.07 D 10 μM 0.23** ±0.03  A = untreated control, B = Ref. 1, C = Seq. ID No. 218b, D = Seq.ID No. 218c, ± = SEM, *p < 0.05, **p < 0.01 in reference to A, +p <0.05, ++p < 0.01 in reference to B. Statistics were calculated using theOrdinary-one-way-ANOVA followed by “Dunnett's” post hoc comparisons.

Conclusion:

Efficient and stable downregulation of target mRNA by gymnotic uptake ofASOs is achieved even in long-term applications. ReNcell CX® cells couldtherefore be used e.g. for experiments addressing recovery of adultneurogenesis as a therapeutic option in patients. The same applies forother indications as shown by A549 experiments.

Taken together, efficient downregulation of TGF-R_(II) is suitableindependently from method of transfer and cell type. Gymnotic uptake ofASOs is the preferred transfer method as in clinical applications theabsence of additional transfection agents suggests high safety forpatients.

Example 2: Determination of Inhibitory Activity of theAntisense-Oligonucleotides Directed to TGF-R_(II) on Protein Level

Western Blot Analysis and Immunocytochemistry was performed to determinewhether reduced TGF-R_(II) mRNA level, mediated by inventiveantisense-oligonucleotides (ASOs) in human lung cancer cells (A549) andhuman neuronal precursor cells (ReNcell CX®) results in a reduction oftarget protein.

Description of Method:

Cells were cultured as described above. For treatment, cells were seededin a 6-well culture dish (Sarstedt #83.3920.300, 80,000 cells/well) and8-well cell culture slide dishes (Sarstedt #94.6140.802, 10,000cells/well) and were incubated overnight at 37° C. and 5% CO₂. Forgymnotic transfer of A549 and ReNcell CX® cell medium was removed andreplaced by fresh full medium (1 ml for 6-well and 0.5 ml for 8-well).Ref. 1 (scrambled control), the respective inventive ASO was then addedin medium at concentrations of 2.5 and 10 μM for protein analysis oftarget downregulation after 72 h in A549 cells and 96 h in ReNcell CX®cells. The cells were lysed and examined by Western Blot as described ingeneral method part. The primary antibody anti-TGF-R_(II) was diluted in0.5% BSA in TBS-T and incubated at 4° C. for 2 days. Afterward membraneswere incubated with the second antibody anti-rabbit IgG HRP-linkeddiluted in 0.5% BSA in TBS-T (1h, RT). Following incubation, blots werewashed with TBS-T, emerged using Luminata™ Forte Western HRP Substrate(Millipore #WBLUF0500) and bands were detected with a luminescent imageanalyzer (ImageQuant™ LAS 4000, GE Healthcare). For housekeepercomparison, the membranes were incubated with HRP-conjugated anti-GAPDH(1:1000 in 0.5% Blotto, 4° C., overnight). Densitometric quantificationwas calculated relative to GAPDH and then normalized to untreatedcontrol with Image Studio™ Lite Software. Procedure forimmunocytochemistry was performed as described in standard protocol. Forverification of target-downregulation anti-TGF-R_(II) was diluted andincubated overnight at 4° C. Cy3 goat-anti-rabbit was used as secondaryantibody. All antibody-dilutions were prepared with Antibody-Diluent(Zytomed® #ZUC025-100). Examination of cells was performed byfluorescence microscopy (Zeiss Axio® Observer.Z1). Images were analyzedwith Image J Software and CorelDRAW® X7 Software.

Results after Gymnotic Transfer:

Western Blot Analysis and immunocytochemistry were used to verify thereduction of TGF-R_(II) protein level. 72 h after gymnotic transfer,TGF-R_(II) protein was significantly reduced using high concentration ofdifferent ASOs according to the invention in comparison to untreatedcontrol in A549 cells (Table 18). Reduced TGF-R_(II) levels were alsoobserved in ReNcell CX® cells (Table 18). For both cell lines, reductionof TGF-R_(II) protein level was shown by Western Blot Analysis.Immunocytochemistry revealed a strong dose-dependent reduction ofTGF-R_(II) protein in both cell lines in comparison to untreated cellsand scrambled control treated cells.

TABLE 18 Densitometric analysis after TGF-R_(II) Western Blot. Reductionof TGF-R_(II) protein after gymnotic transfer with TGF-R_(II) specificASOs in A549 and ReNcell CX ® cells could be observed after 72 h or 96h, respectively. Protein levels were determined relative to housekeepinggene GAPDH using Image Studio ™ Lite Software and were normalized tountreated control. Cell line A549 ReNcell CX Target TGF-R_(II)TGF-R_(II) Time point 72 h, 96 h, n = 3 n = 2 A 1.00 ± 0.00 1.00 ± 0.00B 2.5 μM 0.85 ± 0.13 0.91 ± 0.12 B 10 μM 1.06 ± 0.47 1.23 ± 0.16 C 2.5μM 0.34 ± 0.11 0.59 ± 0.05 C 10 μM 0.39* ± 0.11  0.63 ± 0.17 A =untreated control, B = Ref. 1, C = Seq. ID No. 218b, D = Seq. ID No.218c, F = Seq. ID No. 210q, G = Seq. ID No. 213k, H = Seq. ID No. 143h,I = Seq. ID No. 152h, J = Seq. ID No. 209az, K = Seq. ID No. 209y, ± =SEM, *p < 0.05 in reference to A. Statistics were calculated using theOrdinary-one-way-ANOVA followed by “Dunnett's” post hoc comparisons.

Conclusion:

In addition to target mRNA downregulation, gymnotic transfer of Seq. IDNo. 218b resulted in a reduction of protein level in A549 and ReNcellCX® cells. Staining of TGF-R_(II) revealed a dose-dependent reduction ofTGF-R_(II) protein in both cell lines.

Results after Gymnotic Transfer with Seq. ID No. 218c:

Protein analysis showed a significantly reduced amount of TGF-R_(II) inA549 cells (Table 19). In ReNcell CX® cells gymnotic transfer of 10 μMof ASO Seq. ID No. 218c lead to a reduction of target protein. For bothcell lines, reduction of TGF-R_(II) protein level by gymnotic transferof Seq. ID No. 218c could be detected in comparison to untreated cellsand scrambled control treated cells.

TABLE 19 Results of densitometric analysis after Western Blotting.Reduction of TGF-R_(II) protein after gymnotic transfer withTGF-R_(II)-specific antisense-oligonucletide (ASO) Seq. ID No. 218c inA549 and ReNcell CX ® cells could be observed after 72 h or 96 h,respectively. Protein levels were determined relative tohousekeeping-gene GAPDH using Studio ™ Lite Software and were normalizedto untreated control. Cell line A549 ReNcell CX Target TGF-R_(II)TGF-R_(II) Time point 72 h, 96 h, n = 3 n = 2 A 1.00 ± 0.00 1.00 ± 0.00B 2.5 μM 0.78 ± 0.09 1.27 ± 0.05 B 10 μM 0.79 ± 0.24 1.26 ± 0.22 D 2.5μM 0.68 ± 0.14 1.21 ± 0.28 D 10 μM 0.39* ± 0.07  0.77 ± 0.10 A =untreated control, B = Ref. 1, D = Seq. ID No. 218c, ± = SEM, *p < 0.05in reference to A. Statistics were calculated using theOrdinary-one-way-ANOVA followed by “Dunnett's” post hoc comparisons.

Conclusion:

ASO Seq. ID No. 218c causes downregulation of TGF-R_(II) protein aftergymnotic transfer in A549 and ReNcell CX® cells. This was also verifiedby immunocytochemistry.

Taken together, dose-dependent downregulation of TGF-R_(II) mRNA bygymnotic transfer in A549 and ReNcell CX® cells resulted in adose-dependent reduction of protein levels. Inventive ASOs are potent inprotein target downregulation as demonstrated in A549 and ReNcell CX®cells.

Example 3: Analysis of the Effects of the Antisense-Oligonucleotides tothe Downstream Signaling Pathway of TGF-R_(II)

Functional analyses were performed in human lung cancer cells (A549) andhuman neuronal precursor cells (ReNcell CX®). TGF-β downstream signalingpathway was analyzed, following to an effective downregulation ofTGF-R_(II) mRNA and reduction of protein levels by gymnotic transfer ofthe inventive ASOs. Therefore, mRNA and protein levels of ConnectiveTissue Growth Factor (CTGF), known as downstream-mediator of TGF-β, wereevaluated. In addition, phosphorylation of Smad2 (mothers againstdecapentaphlegic homolog 2) was examined. The phosphorylation of Smad2is a marker for an active TGF-β pathway followed by the upregulation ofthe downstream target gene CTGF.

Description of Method:

Cells were cultured as described before. For treatment, cells wereseeded in a 6-well culture dish (Sarstedt #83.3920.300) (80,000cells/well) and 8-well cell culture slide dishes (Sarstedt #94.6140.802)(10,000 cells/well) and were incubated overnight at 37° C. and 5% CO₂.For gymnotic transfer, A549 and ReNcell CX® cell medium was removed andreplaced by fresh full medium (1 ml for 6-well and 0.5 ml for 8-well).Ref. 1 (Scrambled control), ASO with sequence identification number 218b(Seq. ID No. 218b), No. 218c (Seq.ID No. 218c) was then added in mediumat concentrations of 2.5 and 10 μM and respective analysis was performedafter 72 h in A549 cells and 96 h in ReNcell CX® cells. To evaluateeffects on CTGF mRNA level, real-time RT-PCR was performed as describedbefore. The primer pair for analysis of CTGF was ready-to-use andstandardized. To check for CTGF and pSmad2 protein levels, Western Blotand immunocytochemistry were used as described before. Type and useddilutions of antibodies for respective method are listed in Table 13 and14.

3.1. Results for Seq.ID No.218b

3.1.1 Effects on CTGF mRNA and Protein Level

CTGF mRNA was significantly and dose-dependently reduced after gymnotictransfer with ASO Seq. ID No. 218b in A549 (72 h) and ReNcell CX® (96 h)cells. Downstream-mediator of TGF-β was reduced to 52%±0.02 in ReNcellCX® cells and to 39%±0.03 in A549 cells after gymnotic transfer with 10μM Seq.ID No.218b (Table 20). According to these downregulated CTGF mRNAlevels, a strong reduction of CTGF protein expression was observed inA549 cells (Table 21).

TABLE 20 Dose-dependent and significant downregulation of CTGF mRNAafter gymnotic transfer with Seq. ID No. 218b in A549 and ReNcell CX ®cells. mRNA expression levels were quantified relative to housekeepinggene GNB2L1 using quantitative real-time RT-PCR and normalized tountreated control. Cell line A549 ReNcell CX Target CTGF CTGF Time point72 h, 96 h, n = 3 n = 3 A 1.00 ± 0.08 1.00 ± 0.04 B 2.5 μM 0.87 ± 0.060.97 ± 0.06 B 10 μM 0.80 ± 0.03 0.86 ± 0.17 C 2.5 μM 0.60** ± 0.04 0.66** ± 0.02  C 10 μM 0.39** ± 0.03  0.52** ± 0.02  A = untreatedcontrol, B = Ref. 1, C = Seq. ID No. 218b. ± = SEM, *p < 0.05, **p <0.01 in reference to A. Statistics was calculated using theOrdinary-one-way-ANOVA followed by “Dunnett's” post hoc comparisons.

TABLE 21 Densitometric analysis of CTGF Western Blot. Downregulation ofCTGF protein 72 h after gymnotic transfer with ASO Seq. ID No. 218b inA549 was recognized. Protein levels were determined relative tohousekeeping gene alpha-Tubulin using Studio ™ Lite Software and werenormalized to untreated control. Cell line A549 Target CTGF Time point72 h, n = 1 A 1.00 B 2.5 μM 0.91 B 10 μM 1.31 C 2.5 μM 0.05 C 10 μM0.086 A = untreated control, B = Ref. 1, C = Seq. ID No. 218b.

Conclusion:

Functional inhibition of TGF-β signaling was achieved with gymnotictransfer of Seq. ID No. 218b as shown by downregulation of target CTGFmRNA and reduced CTGF protein levels in A549 and ReNcell CX® cells.

3.1.2 Effects on pSmad2 Protein Level

pSmad2 protein levels were analyzed to proof the CTGF downregulation asa specific result of the ASO-mediated TGF-β signaling inhibition.

Staining against pSmad2 after gymnotic transfer of ASO Seq. ID No. 218bafter 72 h in A549 and 96 h in ReNcell CX® cells showed a dose-dependentinhibition of Smad2 phosphorylation (FIG. 5). In addition, reduction ofpSmad2 expression levels by ASO Seq. ID No. 218b was verified by WesternBlot Analysis in A549 cells (Table 22).

TABLE 22 Densitometric analysis of pSmad2 Western Blot. Downregulationof pSmad2 protein 72 h after gymnotic transfer with ASO Seq. ID No. 218bin A549 was recognized. Protein levels were determined relative tohousekeeping gene GAPDH using Studio ™ Lite Software and normalized tountreated control. Cell line A549 Target pSmad2 Time point 72 h, n = 1 A1.00 B 2.5 μM 1.81 B 10 μM 1.79 C 2.5 μM 0.66 C 10 μM 0.72 A = untreatedcontrol, B = Ref. 1, C = Seq. ID No. 218b.

Conclusion:

Gymnotic transfer of Seq. ID No. 218b in A549 and ReNcell CX® cellsresulted in a dose-dependent inhibition of downstream mediators of TGF-βsignaling. CTGF and phosphorylation of Smad2 was reduced by ASO Seq. IDNo. 218b, both indicating an inhibited TGF-β pathway.

3.2 Results for Seq.ID No. 218c

3.2.1 Effects on CTGF mRNA and pSmad2 Protein Level

Gymnotic transfer of ASO Seq. ID No. 218c downregulates CTGF mRNA inA549 and ReNcell CX® cells (Table 23). Immunocytochemistry againstpSmad2 confirmed an inhibition of TGF-β signaling (FIG. 6). Therefore,downregulation of CTGF mRNA is an direct effect of reduced TGF-βsignaling.

TABLE 23 Significant downregulation of CTGF mRNA was observed in A549and ReNcell CX ® cells. mRNA expression levels were quantified relativeto housekeeping gene GNB2L1 using quantitative real-time RT-PCR andnormalized to untreated controls. Cell line A549 ReNcell CX Target CTGFCTGF Time point 72 h, 96 h, n = 4 n = 3 A 1.00 ± 0.08 1.00 ± 0.10 B 2.5μM 0.97 ± 0.07 0.88 ± 0.08 B 10 μM 0.85 ± 0.06 0.89 ± 0.07 D 2.5 μM0.49** ± 0.05  1.10 ± 0.08 D 10 μM 0.31** ± 0.03  0.82 ± 0.02 A =untreated control, B = Ref. 1, D = Seq. ID No. 218c. ± = SEM, **p < 0.01in reference to A. Statistics was calculated using theOrdinary-one-way-ANOVA followed by “Dunnett's” post hoc comparisons.

Conclusion:

ASO Seq. ID No. 218c was efficient in inhibiting TGF-β signaling afterdownregulation of target TGF-R_(II) mRNA. This was examined bydetermination of downregulated CTGF mRNA and reduced pSmad2 proteinlevels as a marker for TGF-β signaling.

Taken together, inventive ASOs are efficient in mediating a functionalinhibition of TGF-β signaling by downregulation of TGF-R_(II). Thus,inventive ASOs will be beneficial for medical indications in whichelevated TGF-β levels are involved, e.g. neurological disorders,fibrosis and tumor progression.

Example 4: Inhibitory Activity of the Inventive ASOs on Target mRNALevels in TGF-β11 Treated Cells

4.1 Gymnotic Uptake of ASOs in A549 and ReNcell CX® Cells after TGF-β1Pre-Treatment

To analyze inhibitory activity of antisense oligonucleotides (ASOs) inhuman neuronal progenitor cells from cortical brain region (ReNcell CX®)under pathological conditions, cells were pre-treated with TransformingGrowth Factor-β 1 (TGF-β1). From previous studies it is known thatTGF-β1 is found in high concentrations in Cerebrospinal Fluid (CSF) ofall neural disorders e.g. ALS. Therefore, inhibitory efficacy of ASOs onTGFβ-signaling was examined after pre-treatment and in presence withTGF-β 1. A549 cells were used as reference cell line.

Description of Method:

A549 and ReNcell CX® were cultured as described above. For treatmentstudies cells were seeded in a 24-well culture dish (Sarstedt#83.1836.300) (50,000 cells/well) and were incubated overnight at 37° C.and 5% CO₂. For treatment of A549 and ReNcell CX® cells, medium wasremoved and replaced by fresh full medium (0.5 ml for 24-well).Following TGF-β1 (10 ng/ml, PromoCell #C63499) exposition for 48 h,medium was changed, TGF-β1 re-treatment was performed in combinationwith Ref.1 (Scrambled control, 10 μM), ASO Seq. ID No. 218b (10 μM), orASO Seq. ID No. 218c (10 μM) in medium. A549 cells were incubated forfurther 72 h, whereas ReNcell CX® cells were harvested after 96 h.Therefore, cells were washed twice with PBS and subsequently used forRNA isolation (24-well dishes) as described before. Used primer pairsfor real-time RT-PCR are listed in Table 11.

4.1.1 Results for Seq. ID No. 218b

Efficacy in mRNA downregulation of TGF-R_(II) by ASO Seq. ID No. 218bwas not influenced by TGF-β1 pre-incubation in A549 and ReNcell CX®cells (Table 24, FIG. 7). Target mRNA in A549 cells was significantlydownregulated after single treatment (remaining mRNA: 15%±0.05) withASO, but also after treatment in presence of TGF-β1, followingpre-treatment (remaining mRNA: 7%±0.01). In ReNcell CX® cells ASO Seq.ID No. 218b showed similar potency in inhibiting TGF-R_(II) mRNA inabsence of TGF-β1 (25%±0.01) or in presence of TGF-β1, followingpre-treatment of TGF-β1 (17%±0.02).

TABLE 24 In presence of TGF-β1, ASO Seq. ID No. 218b leads to a potentdownregulation of TGF-R_(II) mRNA after gymnotic transfer in A549 andReNcell CX ® cells. mRNA expression levels were quantified relative tohousekeeping gene GNB2L1 using quantitative real-time RT-PCR andnormalized to untreated controls. Target TGF-R_(II) Time point 48 hTGF-β1 −> 72 h/96 h TGF-β1 + ASOs/single treatment Cell line A549ReNcell CX n = 4 n = 3 A 1.00 ± 0.07 1.00 ± 0.11 B 10 μM 0.90 ± 0.170.89 ± 0.26 C 10 μM 0.15** ± 0.05  0.25 ± 0.01 E 10 ng/ml 0.71 ± 0.050.79 ± 0.34 E 10 ng/ml + B 10 μM 0.74 ± 0.05 0.89 ± 0.25 E 10 ng/ml + C10 μM 0.07** ± 0.01  0.27 ± 0.02 A = untreated control, B = Ref. 1, C =Seq. ID No. 218b, E = TGF-β1, ± = SEM, *p < 0.05, **p < 0.01 inreference to A. Statistics was calculated using theOrdinary-one-way-ANOVA followed by “Dunnett's” post hoc comparisons.

Conclusion:

Target mRNA was efficiently downregulated to approx. 20% by gymnoticuptake of inventive ASOs in presence of TGF-β1, following pre-incubationin both tested cell lines.

4.1.2 Results for Seq. ID No. 218c

Downregulation of TGF-R_(II) mRNA by ASO Seq. ID No. 218c was effectivein presence of TGF-β1 in A549 and ReNcell CX® cells (Table 25, FIG. 8).Target mRNA in both tested cell lines was significantly downregulated,regardless of a single treatment with ASO Seq. ID No. 218c or inpresence with TGF-β1.

TABLE 25 In presence of TGF-β1, ASO Seq. ID No. 218c leads to a potentdownregulation of TGF-R_(II) mRNA after gymnotic transfer in A549 andReNcell CX ® cells. mRNA expression levels were quantified relative tohousekeeping gene GNB2L1 using quantitative real-time RT-PCR andnormalized to untreated control. Target TGF-R_(II) Time point 48 hTGF-β1 −> 72 h/96 h TGF-β1 + ASOs/single treatment Cell line A549ReNcell CX n = 2 n = 2 A 1.00 ± 0.12 1.00 ± 0.18 B 10 μM 0.92 ± 0.060.51 ± 0.14 D 10 μM 0.31** ± 0.04  0.05** ± 0.01  E 10 ng/ml 0.68 ± 0.050.88 ± 0.73 E 10 ng/ml + B 10 μM 0.86 ± 0.04 0.45 ± 0.09 E 10 ng/ml + D10 μM 0.16** ± 0.05  0.03** ± 0.01  A = untreated control, B = Ref. 1, D= Seq. ID No. 218c, E = TGF-β1, ± = SEM, *p < 0.05, **p < 0.01 inreference to A. Statistics was calculated using theOrdinary-one-way-ANOVA followed by “Dunnett's” post hoc comparisons.

Conclusion:

Taken together, the inventive ASOs were effective in downregulatingTGF-R_(II) mRNA in presence of TGF-β1, indicating that ASOs arefunctional under pathological conditions.

Example 5: Inhibitory Activity of the Inventive ASOs on Target ProteinLevels in TGF-β11 Treated Cells

To analyze inhibitory activity of antisense oligonucleotides (ASOs) inhuman neuronal progenitor cells from cortical brain region (ReNcell CX®)under pathological conditions, cells were pre-treated with TransformingGrowth Factor-β 1 (TGF-β 1). From previous studies it is known thatTGF-β1 is found in high concentrations in Cerebrospinal Fluid (CSF) ofall neural disorders e.g. ALS. Therefore, inhibitory efficacy of ASOs onTGFβ-signaling was examined after pre-treatment and in presence withTGF-β 1. A549 cells were used as reference cell line.

Description of Method:

Cells were cultured as described before in standard protocol. Fortreatment, cells were seeded in a 6-well culture dish (Sarstedt#83.3920.300) (80,000 cells/well) and 8-well cell culture slide dishes(Sarstedt #94.6140.802) (10,000 cells/well) and were incubated overnightat 37° C. and 5% CO₂. For investigation of gymnotic transfer effects(A549 and ReNcell CX), after pre-incubation with TGF-β1 (Promocell #C-63499), medium was removed and replaced by fresh full medium (1 ml for6-well dishes and 8-well cell culture slide dishes). Followingexposition of TGF-β1 (10 ng/ml, 48 h) medium was changed, TGF-β1 (10ng/ml), Ref.1 (Scrambled control, 10 μM), and inventive ASOs (10 μM) wasadded, in combination and in single treatment, to the cells. A549 cellswere incubated for further 72 h, whereas ReNcell CX® cells wereharvested after 96 h. Therefore, cells were washed twice with PBS andsubsequently used for protein isolation (6-well dishes) followingWestern Blot analysis or immunocytochemical examination of cells (in8-well cell culture slide dishes). Procedures for used techniques wereperformed as described before. Used antibodies and dilutions forrespective methods are listed in Table 13 and 14.

Results after Gymnotic Transfer

Western Blot and immunocytochemical analysis for A549 cells showed thatthe ASOs having Seq. ID No. 218b, Seq. ID No. 218c, Seq. ID No. 210q,Seq. ID No. 213k, Seq. ID No. 143h, Seq. ID No. 152h, Seq. ID No. 209az,Seq. ID No. 209y generate a potent target downregulation in presence ofTGF-β1 (Table 26). Staining of TGF-R_(II) on fixed ReNcell CX® cellsconfirmed the results observed in A549 cells. Tested ASOs revealed astrong target downregulation after single treatment but also in presencewith TGF-β1.

TABLE 26 Densitometric analysis of TGF-R_(II) Western Blot.Downregulation of TGF-R_(II) protein after TGF-β1 pre-incubationfollowed by gymnotic transfer with ASO Seq. ID No. 218b in A549 wasobserved. Protein levels were determined relative to housekeeping geneGAPDH using Studio ™ Lite Software and were then normalized to untreatedcontrol. Target TGF-R_(II) Time point 48 h TGF-β1 −> 72 h TGF-β1 +ASOs/single treatment Cell line A549 n = 1 A 1.00 B 10 μM 1.20 C 10 μM0.31 E 10 ng/ml 2.03 E 10 ng/ml + B 10 μM 1.50 E 10 ng/ml + C 10 μM 0.78A = untreated control, B = Ref. 1, C = Seq. ID No. 218b, E = TGF-β1.

Conclusion:

TGF-β1 pre-incubation followed by gymnotic transfer of Seq. ID No. 218bresulted, in addition to target mRNA downregulation, in a reduction ofprotein level in A549 and ReNcell CX® cells.

5.2 Results of Seq. ID No. 218c after TGF-β11 Pre-Incubation

Western Blot analysis showed a reduced amount of TGF-R_(II) protein inA549 cells (Table 27) after gymnotic transfer for 72 h in comparison tountreated cells and cells treated with scrambled control. Pre-incubationof TGF-β1 followed by gymnotic transfer of tested ASO evoked a reductionin comparison to cells which were pre-treated with TGF-β1 followed bygymnotic transfer with scrambled control. Immunocytochemical examinationof A549 and ReNcell CX® after staining against TGF-R_(II) showed thattested ASO mediated a strong reduction of target protein after gymnotictransfer with or without pre-treatment of TGF-β1.

TABLE 27 Densitometric analysis of TGF-R_(II) Western Blot. Reduction ofTGF-R_(II) protein after TGF-β1 pre-incubation followed by gymnotictransfer with ASO Seq. ID No. 218c in A549 could be detected. Proteinlevels were determined relative to housekeeping gene GAPDH usingStudio ™ Lite Software and were then normalized to untreated control.Target TGF-R_(II) Time point 48 h TGF-β1 −> 72 h TGF-β1 + ASOs/singletreatment Cell line A549 n = 1 A 1.00 B 10 μM 1.10 D 10 μM 0.42 E 10ng/ml 2.03 E 10 ng/ml + B 10 μM 1.50 E 10 ng/ml + D 10 μM 1.16 A =untreated control, B = Ref. 1, D = Seq. ID No. 218c, E = TGF-β1.

Conclusion:

Even after TGF-β1 pre-incubation, gymnotic transfer of Seq. ID No. 218cresults in reduction of TGF-R_(II) protein in A549 and ReNcell CX®cells.

Example 6: Analysis of the Effects of the Inventive ASOs to theDownstream Signaling Pathway of TGF-R_(II) after TGF-β 3-Preincubation

Functional analyses were performed in human lung cancer cells (A549) andhuman neuronal precursor cells (ReNcell CX®). TGF-β1 downstreamsignaling pathway was analyzed, following to an effective downregulationof TGF-R_(II) mRNA and reduction of protein levels by gymnotic transferof the inventive ASOs in presence of TGF-β 1. Therefore, mRNA andprotein levels of Connective Tissue Growth Factor (CTGF), known asdownstream-mediator of TGF-β, were evaluated. In addition,phosphorylation of Smad2 (mothers against decapentaphlegic homolog 2)was examined. The phosphorylation of Smad2 is a marker for an activeTGF-β pathway followed by the upregulation of the downstream target geneCTGF.

Description of Method:

Cells were cultured as described before in standard protocol. Fortreatment, cells were seeded in 24-well culture dishes (Sarstedt#83.1836.300) (50,000 cells/well), 6-well culture dishes (Sarstedt#83.3920.300) (80,000 cells/well) and 8-well cell culture slide dishes(Sarstedt #94.6140.802) (10,000 cells/well) and were incubated overnightat 37° C. and 5% CO₂. For investigation of gymnotic transfer effects(A549 and ReNcell CX® cells), after pre-incubation with TGF-β1, mediumwas removed and replaced by fresh full medium (1 ml for 6-well dishesand 8-well cell culture slide dishes). Following exposition of TGF-β1(10 ng/ml, 48 h) medium was changed, TGF-P1 (10 ng/ml), Ref.1 (Scrambledcontrol, 10 μM), ASO with Seq. ID No. 218b (10 μM), and ASO with Seq. IDNo. 218c (10 μM) was added in combination and in single treatment tocells. A549 cells were incubated for further 72h, whereas ReNcell CX®cells were harvested after 96 h. Therefore, cells were washed twice withPBS and subsequently used for RNA (24-well dishes) and protein isolation(6-well dishes) or immunocytochemical examination of cells (in 8-wellcell culture slide dishes). To evaluate effects on CTGF mRNA level,real-time RT-PCR was performed as described before. The primer pair foranalysis of CTGF was ready-to-use and standardized. To check for CTGFand pSmad2 protein levels, Western Blot and immunocytochemistry wereused as described before. Type and used dilutions of antibodies forrespective method are listed in Table 13 and 14.

6.1. Results for Seq. ID No. 218b

6.1.1 Effects on CTGF mRNA and Protein Levels

CTGF mRNA was downregulated after gymnotic transfer with ASO Seq. ID No.218b in A549 (72 h, 0.52±0.05) and ReNcell CX® (96 h, 0.70±0.25) cells,whereas TGF-31 incubation for 5 days (A549: 48 h+72 h, 6.92±2.32) or 6days (ReNcell CX: 48 h+96 h, 1.60±015) respectively, caused significantupregulation of CTGF mRNA. ASO Seq. ID No. 218b was potent enough toevoke a CTGF mRNA downregulation by blocking TGF-β1 effects in presenceof TGF-β1 (Table 28, FIG. 11). According to observations for mRNAlevels, immunochemical staining against CTGF also confirmed theseobservations for protein levels (FIG. 12).

TABLE 28 Downregulation of CTGF mRNA in presence of TGF-β1 followed bygymnotic transfer with Seq. ID No. 218b in A549 and ReNcell CX ® cells.MRNA expression levels were quantified relative to housekeeping GNB2L1using quantitative real-time RT-PCR normalized to untreated control.Target CTGF Time point 48 h TGF-β1 −> 72 h/96 h TGF-β1 + ASOs/singletreatment Cell line A549 ReNcell CX n = 5 n = 3 A 1.00 ± 0.22  1.00 ±0.04 B 10 μM 0.89 ± 0.19  0.85 ± 0.01 C 10 μM 0.52 ± 0.05  0.70* ± 0.25E 10 ng/ml 6.92* ± 2.32  1.60** ± 0.15 E 10 ng/ml + B 10 μM 8.79** ±2.72  1.71** ± 0.03 E 10 ng/ml + C 10 μM 2.53⁺⁺ ± 0.59   1.19⁺⁺ ± 0.04 A= untreated control, B = Ref. 1, C = Seq. ID No. 218b, E = TGF-β1, ± =SEM, *p < 0.05, **p < 0.01 in reference to A, ⁺⁺p < 0.01 in reference toE + B. Statistics was calculated using the Ordinary-one-way-ANOVAfollowed by “Tukey's” post hoc comparisons.

Conclusion:

In presence of TGF-β1 and following treatment of ASO Seq. ID No. 218bresulted firstly in downregulation of TGF-R_(II) mRNA and secondary inreduced CTGF mRNA and protein levels in A549 and ReNcell CX® cells. Thatindicates that ASO Seq. ID No. 218b is potent enough to be active underhigh TGF-β1 pathological conditions and is able to rescue from TGF-β1mediated effects.

6.1.2 Effects on pSmad2 Protein Level

To verify if CTGF downregulation is a consequence of specific TGF-βsignaling inhibition, mediated by ASO Seq. ID No. 218b in presence ofTGF-β1, pSmad2 protein levels were analyzed.

Staining pSmad2 after TGF-β1 pre-incubation followed by gymnotictransfer of ASO Seq. ID No. 218b with parallel TGF-β1 exposition leadsto an inhibition of Smad2 phosphorylation in both tested cell lines(FIG. 13). In addition, reduced pSmad2 protein levels were verified byWestern Blot Analysis in A549 and ReNcell CX® cells (Table 29).

TABLE 29 Densitometric analysis of pSmad2 Western Blot. Downregulationof pSmad2 protein after gymnotic transfer with ASO Seq. ID No. 218b wasrecognized. Also reversion of TGF-β1 mediated effects by inventive ASOswas found, when combination treatments were compared. Protein levelswere determined relative to housekeeping gene GAPDH using Studio ™ LiteSoftware and were then normalized to untreated control. Target pSmad2Time point 48 h TGF-β1 −> 72 h/96 h TGF-β1 + ASOs/single treatment Cellline A549 ReNcell CX n = 2 n = 2 A 1.00 ± 0.00 1.00 ± 0.00 B 10 μM 1.23± 0.47 0.89 ± 0.22 C 10 μM 0.58 ± 0.08 0.66 ± 0.14 E 10 ng/ml 1.40 ±0.31 1.19 ± 0.61 E 10 ng/ml + B 10 μM 1.27 ± 0.46 2.19 ± 0.76 E 10ng/ml + C 10 μM 0.81 ± 0.31 1.55 ± 0.42 A = untreated control, B = Ref.1, C = Seq. ID No. 218b, E = TGF-β1. Statistics was calculated using theOrdinary-one-way-ANOVA followed by “Dunnett's” post hoc comparisons.

Conclusion:

ASO Seq. ID No. 218b results in a functional inhibition of TGF-βsignaling in A549 and ReNcell CX® cells in presence of TGF-β1, confirmedby reduced phosphorylation of Smad2.

6.2 Results for Seq. ID No. 218c

6.2.1 Effects on CTGF mRNA and Protein Level

Data show CTGF mRNA downregulation after combination treatment with ASOSeq. ID No. 218c and TGF-β1 (A549: 0.86, ReNcell CX®: 0.23) compared tocombination treatment with scrambled control and TGF-β1 (A549: 5.89,ReNcell CX®: 1.25) (Table 30 and FIG. 14). In addition to theseobservations, immunochemical staining of CTGF confirmed prevention ofTGF-β1 mediated effects on protein level by ASO Seq. ID No. 218c (FIG.15).

TABLE 30 CTGF mRNA levels after TGF-β1 pre-incubation followed bygymnotic transfer of Seq. ID No. 218c and parallel TGF- β1 treatment inA549 and ReNcell CX ® cells. Data confirmed effective prevention ofTGF-β1 effects on CTGF mRNA levels by ASO Seq. ID No. 218c. mRNAexpression levels were quantified relative to housekeeping gene GNB2L1using quantitative real-time RT-PCR normalized to untreated controls.Target CTGF Time point 48 h TGF-β1 −> 72 h/96 h TGF-β1 + ASOs/singletreatment Cell line A549 ReNcell CX n = 3 n = 2 A 1.00 ± 0.05 1.00 ±0.03 B 10 μM 0.86 ± 0.11 0.85 ± 0.01 D 10 μM 0.53 ± 0.10 0.17* ± 0.02  E10 ng/ml 4.71 ± 1.76 1.39 ± 0.08 E 10 ng/ml + B 10 μM 5.89* ± 2.16  1.25± 0.44 E 10 ng/ml + D 10 μM 0.86⁺⁺ ± 0.06  0.23*⁺⁺ ± 0.02    A =untreated control, B = Ref. 1, D = Seq. ID No. 218c, E = TGF-β1, ± =SEM, **p < 0.01 in reference to A, ⁺⁺p < 0.01 in reference to E + B.Statistics was calculated using the Ordinary-one-way-ANOVA followed by“Tukey's” post hoc comparisons.

Conclusion:

Data confirmed an effective prevention of TGF-β1 induced effects on CTGFmRNA and protein levels by ASO Seq. ID No. 218c.

6.2.2 Effects on pSmad2 Protein Level

To verify if CTGF downregulation (6.2.1) is a consequence of TGF-β1signaling-inhibition mediated by ASO Seq. ID No. 218c, even in presenceof TGF-β1-preincubation, pSmad2 protein levels were analyzed.

Phosphorylation of Smad2 was induced by TGF-β1 incubation (1.52±0.19),whereas ASO gymnotic transfer mediated a reduction of pSmad2 in A549cells (0.89±0.05). TGF-β1 pre-incubation with following combinationtreatment results in suppression of TGF-β1 effects on phosphorylation ofSmad2 (Western Blot Analysis, Table 31). Immunocytochemistry supportedthe data observed by Western Blot Analysis (FIG. 16).

TABLE 31 Densitometric analysis of pSmad2 Western Blot. Downregulationof pSmad2 protein after gymnotic transfer with ASO Seq. ID No. 218c wasmeasured. Suppression of TGF-β1 mediated effects by inventive ASOs wasshown, when combination treatments were compared. Protein levels weredetermined relative to housekeeping gene GAPDH using Studio ™ LiteSoftware and normalized to untreated controls. Target pSmad2 Time point48 h TGF-β1 −> 72 h TGF-β1 + ASOs/single treatment Cell line A549 n = 2A 1.00 ± 0.00 B 10 μM 1.23 ± 0.27 D 10 μM 0.89 ± 0.05 E 10 ng/ml 1.52 ±0.19 E 10 ng/ml + B 10 μM 1.27 ± 0.29 E 10 ng/ml + D 10 μM 0.93 ± 0.35 A= untreated control, B = Ref. 1, D = Seq. ID No. 218c, E = TGF-β1.Statistics was calculated using the Ordinary-one-way-ANOVA followed by“Dunnett's” post hoc comparisons.

Conclusion:

ASO Seq. ID No. 218c is efficiently inhibiting TGF-β signaling afterTGF-β1 pre-incubation followed by ASO gymnotic transfer. This was shownby examination of downstream pSmad2 protein levels.

Taken together, inventive ASOs are extraordinary capable in mediating afunctional inhibition of TGF-β signaling in presence of pathological,high TGF-β1 levels by efficiently downregulating TGF-R_(II) mRNA. Thus,inventive ASOs will be beneficial in medical indications in whichelevated TGF-β levels are involved, e.g. neurological disorders,fibrosis, tumor progression and others.

Example 7: Determination of Prophylactic Activity of theAntisense-Oligonucleotides on mRNA Level (TGF-β11 Post-Treatment)

To analyze prophylactic activity of antisense-oligonucleotides (ASOs) inhuman neuronal progenitor cells from cortical brain region (ReNcellCX®), ASOs were transferred to cells by gymnotic uptake followingTransforming Growth Factor-31 (TGF-β 1) treatment.

Description of Method:

A549 and ReNcell CX® cells were cultured as described above. Forprophylactic treatment studies, cells were seeded in a 24-well culturedish (Sarstedt #83.1836.300) (50,000 cells/well) and were incubatedovernight at 37° C. and 5% CO₂. Afterwards, Ref.1 (Scrambled control, 10μM) or ASO with Seq. ID No. 218b (10 μM) were added to media for 72 h(A549) or 96 h (ReNcell CX®). Following incubation time after gymnotictransfer, TGF-β 1 (10 ng/ml, Promocell #C63499) was added, withoutmedium replacement, to the cells for further 48 h. For harvesting, cellswere washed twice with PBS and subsequently used for RNA isolation(24-well dishes) following mRNA analysis by real-time RT-PCR.Ready-to-use and standardized primer pairs for real-time RT-PCR wereused and mixed with the respective ready-to-use Mastermix solution(SsoAdvanced™ Universial SYBR® Green Supermix (BioRad #172-5271)according to manufacturer's instructions (BioRad Prime PCR Quick Guide).Methods were performed as described above.

7.1 Results for Seq. ID No. 218b

Efficacy in TGF-R_(II) mRNA downregulation by ASO Seq. ID No. 218b wasnot influenced by TGF-β1 post-incubation in A549 and ReNcell CX® cells(Table 32). Significant decrease of target mRNA in ReNcell CX® cells wasshown after single treatment (0.33*±0.11) with ASO Seq. ID No. 218b. ASOgymnotic transfer with post-treatment of TGF-β1, strongly reduced thetarget TGF-R_(II) mRNA. In A549 cells, Seq. ID No. 218b showed similarpotency in inhibiting TGF-R_(II) mRNA in single (0.25±0.07) orcombination treatment with post-incubation of TGF-β1 (0.24±0.06).

TABLE 32 Downregulation of TGF-R_(II) mRNA after gymnotic transferfollowing TGF-β1 treatment of inventive ASO in A549 and ReNcell CX ®cells. mRNA expression levels were quantified relative to housekeepinggene GNB2L1 using quantitative real-time RT-PCR normalized to untreatedcontrol. Target TGF-R_(II) Time point 72 h/96 h ASOs −> 48 h TGF-β1 Cellline A549 ReNcell CX n = 3 n = 3 A 1.00 ± 0.44 1.00 ± 0.19 B 10 μM 0.95± 0.22 1.42 ± 0.14 C 10 μM 0.25 ± 0.07 0.33* ± 0.11  E 10 ng/ml 1.96 ±0.16 1.42 ± 0.08 E 10 ng/ml + B 10 μM 1.14 ± 0.39 1.25 ± 0.14 E 10ng/ml + C 10 μM 0.24⁺⁺ ± 0.06  0.56⁺⁺ ± 0.10  A = untreated control, B =Ref. 1, C = Seq. ID No. 218b, E = TGF-β1. ± = SEM, *p < 0.05 inreference to A, ⁺⁺p < 0.01 in reference to E + B. Statistics wascalculated using the Ordinary-one-way-ANOVA followed by “Tukey's” posthoc comparisons.

Conclusion:

Gymnotic uptake of ASO Seq. ID No. 218b followed by TGF-β1post-incubation was effective in target TGF-R_(II) mRNA downregulation,indicating that ASO Seq. ID No. 218b is feasible for prophylactictreatment in medical indications.

Example 8: Determination of Inhibitory Activity of the Inventive ASOs onProtein Level Following TGF-β1 Treatment

To analyze prophylactic activity of inventive ASOs in human neuronalprogenitor cells from cortical brain region (ReNcell CX®), ASOs weretransferred to cells by gymnotic uptake following TGF-β1 treatment.

Description of Method:

Cells were cultured as described before in standard protocol. Fortreatment cells were seeded in 8-well cell culture slide dishes(Sarstedt #94.6140.802) (10,000 cells/well) and were incubated overnightat 37° C. and 5% CO₂. Afterwards, Ref.1 (Scrambled control, 10 μM) orASO sequence identification number 218b (Seq. ID No. 218b, 10 μM) wereadded to media for 72 h (A549) or 96 h (ReNcell CX®). Following gymnotictransfer TGF-β1 (10 ng/ml, Promocell #C63499) was added, without mediumreplacement, to the cells for further 48 h. For harvesting, cells werewashed twice with PBS and subsequently used for immunocytochemicalanalysis. Procedure was performed as described before. Used antibodiesand dilutions for respective methods are listed in Table 13 and 14.

8.1 Results of TGF-R_(II) Protein Reduction after Gymnotic Transfer withSeq. ID No. 218b Following TGF-β1 Treatment

Immunocytochemical analysis against TGF-R_(II) for A549 and ReNcell CX®cells showed that ASO Seq. ID No. 218b generates potent TGF-R_(II) mRNAtarget downregulation after following TGF-β1 treatment (FIG. 17).

Conclusion:

Gymnotic transfer of ASO Seq. ID No. 218b following TGF-β1 treatmentresulted in target mRNA downregulation, as well as a strong reduction ofTGF-R_(II) protein level in A549 and ReNcell CX® cells.

Taken together, efficacy of downregulating TGF-R_(II) protein mediatedby ASO Seq. ID No. 218b in combination with post-treatment of TGF-β1 wasstill given, concluding that the inventive ASOs are effective forprophylactic applications.

Example 9: ASO Treatment Effects on Downstream Signaling Pathway ofTGF-R_(II) Following TGF-β1 Treatment

Efficacy of inventive ASOs in mediating an inhibition of TGF-(3signaling was evaluated for TGF-β1 treatment followed gymnotic transferin human lung cancer cells (A549) and human neuronal precursor cells(ReNcell CX®). Therefore, downstream molecules of TGF-β signaling, Smad3(mothers against decapentaphlegic homolog 3) and Connective TissueGrowth factor (CTGF), were analyzed.

Description of Method:

Cells were cultured as described before in standard protocol. Fortreatment, cells were seeded in 24-well culture dishes (Sarstedt#83.1836.300) (50,000 cells/well) and 8-well cell culture slide dishes(Sarstedt #94.6140.802) (10,000 cells/well) and were incubated overnightat 37° C. and 5% CO₂. Afterwards, Ref.1 (Scrambled control, 10 μM) orASO Seq. ID No. 218b (10 μM) were added to media for 72 h (A549) or 96 h(ReNcell CX®). Following gymnotic transfer, TGF-β1 (10 ng/ml, Promocell#C63499) was added without medium replacement for further 48 h. Forharvesting, cells were washed twice with PBS and subsequently used forRNA isolation (24-well dishes) or immunocytochemical examination ofcells (in 8-well cell culture slide dishes). To evaluate effects on CTGFmRNA level, real-time RT-PCR was performed as described before. Theprimer pair for analysis of CTGF was ready-to-use and standardized. Todetermine pSmad3 protein levels, immunocytochemistry was used asdescribed before. Type and used dilutions of antibodies for respectivemethod are listed in Table 13 and 14.

9.1. Results for Seq. ID No. 218b

9.1.1 Effects on CTGF mRNA and pSmad3 Protein Level

CTGF mRNA was reduced after gymnotic transfer with ASO Seq. ID No. 218bin A549 (5 days: 0.67±0.02) and ReNcell CX® (6 days: 0.70±0.02) cells.Adding TGF-β1 after 72 h or 96 h respectively, cells react with anincrease of CTGF mRNA, but in comparison to gymnotic transfer ofscrambled control following TGF-β1 treatment, induction of CTGF mRNA wasstrongly reduced (Table 33). To verify if CTGF mRNA downregulation was aconsequence of TGF-β signaling inhibition, mediated by ASO Seq. ID No.218b, also after followed TGF-β1 treatment, pSmad3 protein levels wereexamined. FIG. 18 demonstrates that TGF-β signaling was in fact blockedby gymnotic transfer of ASO Seq. ID No. 218b in A549 (FIG. 18 A) andReNcell CX® cells (FIG. 18 B). This effect was also present aftergymnotic transfer of tested ASO following TGF-β1 treatment.

TABLE 33 Downregulation of CTGF mRNA after gymnotic transfer of ASO Seq.ID No. 218b followed by TGF-β1 treatment in A549 and ReNcell CX ® cells.Quantification of mRNA expression levels were performed relative tohousekeeping gene GNB2L1 using quantitative real-time RT-PCR and thennormalized to untreated control. Target CTGF Time point 72 h/96 h ASOs−> +/− 48 h TGF-β1 Cell line A549 ReNcell CX n = 3 n = 3 A 1.00 ± 0.131.00 ± 0.09 B 10 μM 0.80 ± 0.03 1.07 ± 0.07 C 10 μM 0.67 ± 0.02 0.70 ±0.02 E 10 ng/ml 4.54** ± 0.68  1.56* ± 0.08  E 10 ng/ml + B 10 μM 4.07**± 0.38  1.62* ± 0.09  E 10 ng/ml + C 10 μM 1.90⁺ ± 0.03  0.97⁺⁺ ± 0.10 A = untreated control, B = Ref. 1, C = Seq. ID No. 218b, E = TGF-β1. ± =SEM, *p < 0.05, **p < 0.01 in reference to A, ⁺p < 0.05, ⁺⁺p < 0.01 inreference to E + B. Statistics was calculated using theOrdinary-one-way-ANOVA followed by “Tukey's” post hoc comparisons.

Conclusion:

Gymnotic transfer of ASO Seq. ID No. 218b resulted in downregulation ofTGF-R_(II) mRNA and protein, as well as in reduced CTGF mRNA and pSmad3protein levels in A549 and ReNcell CX® cells, independently of TGF-β1treatment.

That indicates that ASO Seq. ID No. 218b is potent enough to be alsoactive under prophylactic conditions to resume or reduce ongoing TGF-β1mediated effects.

Example 10: Analysis of Potential Proinflammatory and ToxicologicalEffects of Antisense-Oligonucleotides

10.1 Peripheral Blood Mononuclear Cell (PBMC) Assay

To analyze antisense-oligonucleotide (ASO) for immunostimulatoryproperties, peripheral blood mononuclear cells (PBMCs) were incubatedwith control ASOs and test compounds followed by ELISAs for IFNα andTGFα.

Description of Method:

PBMCs were isolated from buffy coats corresponding to 500 ml full bloodtransfusion units. Each unit was obtained from healthy volunteers andglucose-citrate was used as an anti-agglutinant. The buffy coat wasprepared and delivered by the Blood Bank Suhl on the Institute forTransfusion Medicine, Germany. Each blood donation was monitored for HIVantibody, HCV antibody, HBs antigen, TPHA, HIV RNA, and SPGT (ALAT).Only blood samples tested negative for infectious agents and with anormal SPGT value were used for leukocyte and erythrocyte separation bylow-speed centrifugation. The isolation of PBMCs was performed about 40h following blood donation by gradient centrifugation usingFicoll-Histopague® 1077 (Heraeus™ Multifuge™ 3 SR). For IFNα assay,PBMCs were seeded at 100,000 cells/96-well in 100 μl complete mediumplus additives (RPM11640, +L-Glu, +10% FCS, +PHA-P (5 μg/ml), +IL-3 (10μg/ml)) and test compounds (5 μl) were added for direct incubation (24h, 37° C., 5% CO₂). For TNFα assay, PBMCs were seeded at 100,000cells/96-well in 100 μl complete medium w/o additives (RPM11640, +L-Glu,+10% FCS) and test compounds (5 μl) were added for direct incubation (24h, 37° C., 5% CO₂). ELISA (duplicate measurement out of pooledsupernatants, 20 μl) for huIFNα (eBioscience, #BMS2161NSTCE) wasperformed according to the manufacturer's protocol. ELISA (duplicatemeasurement out of pooled supernatants, 20 μl) for huTNFα (eBioscience,#BMS2231NSTCE) was performed according to the manufacturer's protocol.

Results:

There was no immunostimulatory effect of ASO treatment on PBMCsindicated by no detectable IFNα (Table 34) and TNFα (Table 35) secretionupon ASO incubation. Assay functionality is proven by theimmunostimulatory effect of immunostimulatory, cholesterol-conjugatedsiRNA (XD-01024; IFNα) and polyinosinic:polycytidylic acid (poly I:C;TNFα; InvivoGen # tlrl-pic) which is a synthetic analog ofdouble-stranded RNA, binds to TLR3 and stimulates the immune system.

TABLE 34 IFNα response to inventive ASO exposure: shows the IFNαresponse of PBMCs upon ASO incubation. Quantification of expressionlevels were determined to positive controls (ODN2216 [class A CpGoligonucleotide; recognized by TLR9 and leading to strongimmunostimulatory effects; InvivoGen tlrl-2216], poly I:C, XD-01024)using ELISA assay. Mean of duplicates [pg/ml] Test candidate Donor 1Donor 2 mock −0.084 0.720 Seq. ID No. 209y −0.061 −0.039 Seq. ID No.209t −0.308 −0.520 Seq. ID No. 209v −0.191 −1.252 Seq. ID No. 218b−0.001 −0.093 Seq. ID No. 218m −0.140 −0.163 Seq. ID No. 218q −0.7550.005 Seq. ID No. 218c −0.852 −0.805 Seq. ID No. 218t −0.469 0.450ODN2216 0.300 1.311 poly I:C −1.378 2.053 XD-01024 13.961 26.821 Allvalues except positive control (XD-01024) below limit of quantification

TABLE 35 TNFα response to inventive ASO exposure: Quantification ofexpression levels were determined to control candidates (ODN2216, polyI:C, XD-01024) using ELISA assay. Mean of duplicates [pg/ml] Testcandidate Donor 1 Donor 2 mock 0.647 −0.137 Seq. ID No. 209y 2.397−0.117 Seq. ID No. 209t 0.734 0.193 Seq. ID No. 209v 0.360 0.063 Seq. IDNo. 218b 0.670 0.183 Seq. ID No. 218m 0.594 0.519 Seq. ID No. 218q 0.0490.194 Seq. ID No. 218c −0.212 0.029 Seq. ID No. 218t 0.593 0.758 ODN22160.085 0.894 poly I:C 115.026 102.042 XD-01024 1.188 1.418 All valuesexcept positive control (poly I:C) below limit of quantification

10. 2 In Vivo Toxicology of Inventive Antisense-Oligonucleotides

To analyze antisense-oligonucleotides (ASOs) for toxicologicalproperties, C57/Bl6N mice received three intravenous ASO injections, andfollowing sacrification, transaminase levels within serum, liver andkidney were examined.

Description of Method:

Female C57/Bl6N mice at the age of 6 weeks were treated with testcompounds (Seq. ID No. 218b, Seq. ID No. 218c) for seven days. ASOs (200μl, 15 mg/kg/BW) were injected intravenously on day one, two, and threeof the treatment period. Body weight development (Seq. ID No. 218c) wasmonitored on every consecutive day and on day four serum was collectedfrom the vena fascicularis. On day eight the animals were sacrificed(CO₂) and serum from the vena cava, the liver (pieces of =50 mg), thekidneys, and the lung were collected for mRNA and transaminasequantification. TGF-R_(II) mRNA levels were determined in liver, kidney,and lung lysate by bDNA assay (QuantiGene® kit, Panomics/Affimetrix).Aspartate transaminase (ASP) and alanine transaminase (ALT) weremeasured on Cobas Integra® 400 from 1:10 diluted serum.

TABLE 36 Serum expression levels of alanine transaminase and aspartatetransaminase of C57/BI6N mice following repeated ASO iv injection.Quantification of expression levels was achieved by comparing to theexpression levels of saline-treated animals. Serum transaminases [U/L] 3days post injection 7 days post injection Test compound ALT AST ALT ASTSeq. ID No. 209ax 13.87 ± 1.44 47.33 ± 15.88  64.91 ± 21.01 108.99 ±13.56  Seq. ID No. 143h 13.68 ± 3.33 53.50 ± 6.99  12.47 ± 1.64 33.35 ±8.17  Seq. ID No. 152h 16.66 ± 6.29 67.23 ± 29.91 17.49 ± 2.81 45.75 ±17.14 Seq. ID No. 209ay 18.29 ± 6.37 69.96 ± 35.44 287.29 ± 65.39 273.45± 101.33 Seq. ID No. 210q 11.70 ± 3.80 36.44 ± 5.36  11.11 ± 6.31 40.81± 13.32 Seq. ID No. 218b 19.60 ± 8.62 67.61 ± 42.75 18.38 ± 4.60 48.91 ±17.86 Seq. ID No. 213k 13.59 ± 3.28 54.47 ± 36.15  96.00 ± 46.74 89.12 ±21.82 Saline  9.52 ± 9.21 67.18 ± 28.60  9.99 ± 2.29 28.29 ± 2.23  ± =SEM.

TABLE 37 Expression levels of TGF-R_(II) within liver, kidney, and lungtissue of C57/BI6N mice following repeated ASO iv injection.Quantification of expression levels was achieved by comparing to theexpression levels of saline-treated animals. TGF-RII mRNA/GAPDH mRNAexpression Test compound Liver Kidney Lung Seq. ID No. 209ax 0.64 ± 0.031.31 ± 0.11 13.25 ± 0.67 Seq. ID No. 143h 0.26 ± 0.02 0.65 ± 0.22 11.10± 0.11 Seq. ID No. 152h 0.58 ± 0.10 0.87 ± 0.17 13.42 ± 0.69 Seq. ID No.209ay 0.62 ± 0.06 1.30 ± 0.10 13.93 ± 0.57 Seq. ID No. 210q 0.39 ± 0.060.83 ± 0.15 13.53 ± 1.23 Seq. ID No. 218b 0.72 ± 0.08 0.97 ± 0.06 15.63± 1.45 Seq. ID No. 213k 0.42 ± 0.01 1.20 ± 0.04 14.44 ± 1.03 Saline 0.66± 0.04 1.10 ± 0.08 15.14 ± 0.65 ± = SEM.

TABLE 38 Serum expression levels of alanine transaminase and aspartatetransaminase of C57/BI6N mice following repeated ASO iv injection.Quantification of expression levels was achieved by comparing to theexpression levels of saline-treated animals. Serum transaminases [U/L]Test 3 days post injection 7 days post injection compound ALT AST ALTAST Seq. ID No. 24.63 ± 2.10 51.87 ± 5.99  18.10 ± 4.01 39.99 ± 2.09218c Saline 28.68 ± 3.23 79.95 ± 30.24 14.52 ± 4.89 36.08 ± 3.32 ± =SEM.

TABLE 39 Expression levels of TGF-R_(II) within liver and kidney tissueof C57/BI6N mice following repeated ASO iv injection. Quantification ofexpression levels was achieved by comparing to the expression levels ofsaline-treated animals. TGF-RII mRNA/GAPDH mRNA expression Test compoundLiver Kidney Seq. ID No. 218c 0.21 ± 0.03 0.16 ± 0.02 Saline 0.35 ± 0.050.24 ± 0.03 ± = SEM.

TABLE 40 Body weight development during the 7-day ASO treatmentparadigm. Body weight gain was quantified compared to body weight on day0, which was set to 100%. Body weight development [%] Test compound Day0 Day 1 Day 2 Day 3 Day 4 Day 7 Seq. ID No. 100% 99%  99%  99% 102% 104%218c Saline 100% 99% 100% 100% 101% 103%

Conclusion:

There were no proinflammatory or toxic effects of relevant inventiveASOs on PBMCs or C57/Bl6N mice. Therefore, ASO treatment targetingTGF-R_(II) reflects a safe method to treat a variety of TGF-B associateddisorders.

Example 11: Determination of Intracerebroventricular Infusion ofInventive ASOs on TGF-β Induced Neural Stem Inhibition and NeuralProgenitor Cell Proliferation In Vivo

The goal of the present study was to evaluate the potential of inventiveASOs against TGF-R_(II) i) to prevent and ii) to treat the TGF-β1induced effects on neural stem and progenitor cell proliferation invivo.

Description of Method:

11.1 Prevention of TGF-β1 Associated Downregulation of Neurogenesis

Two-month-old female Fischer-344 rats (n=32) receivedintracerebroventricular infusions via osmotic minipumps (Model 2002,Alzet) connected to stainless steel cannulas. The surgical implantationof the minipumps was performed under deep anesthesia using intramuscularinjections. Animals were infused with inventive ASOs according to theinvention (1.64 mM concentration present in the pump), scrambled ASO(1.64 mM concentration present in the pump) or aCSF (artificialcerebrospinal fluid) for 7 days. At day 8, pumps were changed and theanimals were infused with either i) aCSF, ii) TGF-β1 (500 ng/ml presentin the pump), iii) TGF-β1 (500 ng/ml present in the pump) plus scrambledASO (1.64 mM concentration present in the pump), or iv) TGF-β1 (500ng/ml present in the pump) plus inventive ASO (1.64 mM concentrationpresent in the pump) for 14 days. At the end of the infusion-period allanimals were transcardially perfused with 4% paraformaldehyde. Thebrains were analyzed for cannula tract localization and animals withincorrect cannula placement were excluded from the analysis. During thelast 24 hours of the pump period, the animals received anintraperitoneal injection of 200 mg/kg bromo-deoxyuridine (BrdU).

The tissue was processed for chromogenic immunodetection ofBrdU-positive cells in 40 μm sagital sections. BrdU positive cells werecounted within three 50 μm×50 pm counting frames per section located atthe lowest, middle and upper part of the subventricular zone. Positiveprofiles that intersected the uppermost focal plane (exclusion plane) orthe lateral exclusion boundaries of the counting frame were not counted.For hippocampal analysis, the volume of the hippocampus was determinedand all positive cells within and adjacent to the boundaries werecounted. The total counts of positive profiles were multiplied by theratio of reference volume to sampling volume in order to obtain theestimated number of BrdU-positive cells for each structure. Allextrapolations were calculated for one cerebral hemisphere and should bedoubled to represent the total brain values. Data are presented as meanvalues±standard deviations (SD). Statistical analysis was performedusing the unpaired, two-sided t-test comparison -Student's t-testbetween the TGF-β1 treated and control groups (GraphPad Prism 4software, USA). The significance level was assumed at p<0.05.

11.2 Treatment of TGF-β1 Associated Down-Regulation of Neurogenesis

Animals received either aCSF or recombinant human TGF-ß1 (500 ng/mlpresent in pump) at a flow rate of 0.5 μl per hour for 14 days. After 14days, pumps were changed and the animals were infused with either i)aCSF, ii) recombinant human TGF-B1 (500 ng/ml present in pump) orco-infused with iii) inventive ASO (1.64 mM concentration present in thepump) plus recombinant human TGF-B1 (500 ng/ml present in pump) or iv)scrambled ASO (1.64 mM concentration present in the pump) plusrecombinant human TGF-B1 (500 ng/ml present in pump). At the end of theinfusion-period all animals were transcardially perfused with 4%paraformaldehyde. The brains were analyzed for cannula tractlocalization and animals with incorrect cannula placement were excludedfrom the analysis. During the last 24 hours of the pump period, theanimals received an intraperitoneal injection of 200 mg/kgbromo-deoxyuridine (BrdU).

Histological analysis was done as described above (11.1).

Results:

The treatment with ASO of Seq. ID No. 143aj, Seq. ID No. 143h and Seq.ID No. 210q specifically and partially reduced the effect of TGF-β1 oncell proliferation in the hippocampus and in the ventricle wall.Treatment with an inventive ASO specifically and partially rescues fromthe inhibitory effect of TGF-β1 on neurogenesis.

Conclusion:

The ASOs of the present invention demonstrating cross-reactivity withrodents induce neurogenesis in this in vivo experiment. The ASOs of thepresent invention demonstrating no cross-reactivity, exert mostly evenmore potential effects in in vitro experiments. As a result, it isassumed that these inventive ASOs are also more effective in in vivo setups for non-human primates and humans and therefore act as a highlypotent medication for preventing or treating TGF-B1 induced inhibitionof neural stem and progenitor proliferation.

Example 12: Analysis of the Effect of the InventiveAntisense-Oligonucleotides on Proliferation and Specific Markers ofHuman Neural Progenitor Cells

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative lethaldisorder with no effective treatment so far. The current moleculargenetic campaign is increasingly elucidating the molecular pathogenesisof this fatal disease, from previous studies it is known that TGF-β isfound in high concentrations in Cerebrospinal Fluid (CSF) of ALSpatients. These high levels of circulating TGF-β are known to promotestem cell quiescence and therefore cause inhibition of adultneurogenesis within the subventricular zone (SVZ) of the brain. Thus,regeneration of degenerating neurons seems to be prevented by anenhanced TGF-β signaling.

To figure out if selective inhibition of TGF-β signaling mediated by theinventive antisense-oligonucleotides might allow reactivation of adultneurogenesis, evidence of TGF-β mediated cell cycle arrest has to beproofed.

Description of Methods:

Cell Cycle Arrest Studies:

Cells were cultured as described before in standard protocol. Forexperiments, cells were seeded in 24-well culture dishes (Sarstedt#83.1836.300) (30,000 cells/well) and were incubated overnight at 37° C.and 5% CO₂. For determination of TGF-β1 mediated effects on cell cycleunder proliferative (+EGF/FGF) (Millipore: EGF #GF144, bFGF #GF003) ordifferentiating (−EGF/FGF) conditions, cells were treated for 4 d withTGF-β1 (PromoCell #C63499, 10 or 50 ng/ml) after removing andreplacement of respective medium. At day 4 medium was refreshed andTGF-β1 treatment was repeated until day 7. On day 7, cells wereharvested by washing twice with PBS and subsequently used for RNA(24-well dishes) isolation as described above. For evaluatingTGF-β1-mediated effects on cell cycle by real-time RT-PCR, mRNA ofproliferation marker Ki67, tumor suppressor gene p53, cyclin-dependentkinase inhibitor 1 (p21) and of neurogenesis marker Doublecortin (DCX)were analyzed. Respective primer pairs are listed in Table 11.

mRNA Analysis for Effects of ASO Seq. ID No. 218b on Human NeuralProgenitor Cells:

Cells were cultured as described before in standard protocol. Forexperiments, cells were seeded in 24-well culture dishes (Sarstedt#83.1836.300) (30,000 cells/well) and were incubated overnight at 37° C.and 5% CO₂. For present experiments, cell medium was changed and Ref.1(Scrambled control, 2.5 and 10 μM), ASO with Seq. ID No. 218b (2.5 and10 μM) or TGF-β1 (10 ng/ml, Promocell #C63499) were added to cells for96 h. After incubation time, medium was changed once more and furthertreatment was performed for further 96 h. After 8 days of treatmentcells were harvested. Cells were washed twice with PBS and subsequentlyused for RNA (24-well dishes) isolation. To evaluate effects onprogenitor cells, Nestin (early neuronal marker), Sox2 (early neuronalmarker), DCX (indicator of neurogenesis) and Ki67 (proliferation marker)mRNA levels were determined by real-time RT-PCR as described before.Respective primer pairs are listed in Table 11.

Proliferative and Differentiating Effects of TGFR_(II) Specific ASOs byGymnotic Transfer on ReNcell CX® Cells:

The next goal was to investigate, whether TGF-R_(II) specific ASOinfluence the proliferation of ReNcell CX® cells. Therefore, cells werecultured as described before and seeded in 24-well culture dishes(Sarstedt #83.1836.300) (30,000 cells/well) or 8-well cell culture slidedishes (Sarstedt #94.6140.802) (10,000 cells/well) and were incubatedovernight at 37° C. and 5% CO₂. For obtaining a proliferation curve,cells were treated after medium change for 72 h with Ref.1 (Scrambledcontrol, 2.5 and 10 μM) and with ASO Seq. ID No. 218b (2.5 and 10 μM).After incubation time, medium change and treatment was repeated twotimes. After collecting supernatant, remaining cells were harvested from24-well dishes for determination of cell number. For this purpose,remaining cells were washed with PBS (2×), treated with accutase (500μl/well) and incubated for 5 min at 37° C. Afterwards 500 μl medium wereadded and cell number was determined using Luna FL™ Automated CellCounter Fluorescence and Bright Field (Biozym, #872040) according tomanufacturer's instructions. Briefly, 18 μl of the cell suspension wereadded to 2 μl of acridine orange/propidium iodide assay viability kit(Biozym #872045). After 1 min of settling, 10 μl were added onto CellCounting Slide (Biozym #872011), cells were counted and calculated intotal cells/ml and percentage of alive cells compared to dead cells.After gymnotic transfer of Ref.1 (10 μM), Seq. ID No. 218b (10 μM) andcorresponding treatment of TGF-β1 (10 ng/ml) for 8 days, cells of 8-wellcell culture slide dishes were fixed and stained with an antibodyagainst Ki67. For investigating differentiation ability of ReNcell CX®cells after gymnotic transfer, other 8-well cell culture slide disheswere treated with Ref.1 (10 μM), Seq. ID No. 218b (10 μM) andcorresponding treatment of TGF-β1 (10 ng/ml) for 96 h underproliferative conditions (+EGF/FGF). Afterwards, one part of the cellswas treated for further 96 h under proliferative conditions whereas theother part of cells was treated and hold under differentiatingconditions (−EGF/FGF). Following staining of cells, Neurofilament N(NeuN) and 111-Tubulin expression levels were determined by fluorescencemicroscopy. Protocol for harvesting, fixing and staining cells wasdescribed above and respective antibody dilutions are listed in Table14.

mRNA Analysis of Markers for Proliferation and Neuroqenesis afterGymnotic Transfer Following TGF-β1 Pre-Incubation:

Cells were cultured as described before in standard protocol. Forexperiments cells were seeded in 24-well culture dishes (Sarstedt#83.1836.300) (30,000 cells/well) and incubated overnight at 37° C. and5% CO₂. For inducing cell cycle arrest, ReNcell CX® cells were treatedwith TGF-β1 for 4 days. Afterwards medium was changed and TGF-β1 (10ng/ml) was added freshly. One day 8 medium was changed on more time, andgymnotic transfer was performed for 96 h by adding Ref.1 (10 μM), Seq.ID No. 218b (10 μM) in combination with TGF-31 (10 ng/ml). Cells wereharvested after incubation by washing twice with PBS. Following RNAisolation and mRNA analysis by real-time RT-PCR were performed asdescribed.

12.1.1 Mediation of Cell Cycle Arrest by TGF-β1 in Human NeuralProgenitor Cells

Detection of stem cell quiescence markers showed that TGF-β1 mediatescell cycle arrest 7 days after exposure of cells. Proliferation markerKi67 mRNA expression was dose-dependently reduced. Also mRNA expressionof tumor suppressor gene p53 was downregulated correlating to TGF-β1concentration. In contrast, cyclin-dependent kinase inhibitor 1 (p21)was significantly upregulated by TGF-β1. In summary these resultsindicate stem cell quiescence induced by TGF-β1. Interestingly, DCX, amarker for neurogenesis, was strongly reduced by TGF-β1 (Table 41).

TABLE 41 mRNA expression of Ki67, p27, p21, and DCX 7 days after TGF-β1treatment in ReNcell CX ® cells. mRNA expression levels were determinedrelative to housekeeping gene GNB2L1 using quantitative real-time RT-PCRand then normalized to untreated control. Cell line ReNcell CX mRNAlevels 7 days after TGF-β1 exposure Target Ki67 p53 p21 DCX n = 3 n = 3n = 3 n = 3 A + EGF/ 1.00 ± 0.38 1.00 ± 0.38 1.00 ± 0.25 1.00 ± 0.49 FGFE 10 ng/ml + 0.67 ± 0.20 0.66 ± 0.18 1.90* ± 0.22  0.37 ± 0.06 EGF/FGF E50 ng/ml + 0.43 ± 0.09 0.42 ± 0.06 1.45 ± 0.16 0.16 ± 0.01 EGF/FGF A −EGF/ 1.00 ± 0.15 1.00 ± 0.13 1.00 ± 0.14 1.00 ± 0.31 FGF E 10 ng/ml −0.87 ± 0.08 0.97 ± 0.10 1.00 ± 0.04 0.72 ± 0.14 EGF/FGF E 50 ng/ml −0.93 ± 0.11 0.93 ± 0.09 0.90 ± 0.09 0.71 ± 0.24 EGF/FGF A = untreatedcontrol, E = TGF-β1. ± = SEM, *p < 0.05 in reference to A. Statisticswas calculated using the Ordinary-one-way-ANOVA followed by “Tukey's”multiple post hoc comparison.

Conclusion

Proliferation of ReNcell CX® Cells was Blocked by TGF-β 1.

12.1.2 Results of Antisense-Oligonucleotide Effects on Markers of HumanNeuronal Stem Cells

To figure out the effect of ASO Seq. ID No. 218b on stem cell markers, 8days after repeated gymnotic transfer (2×96 h) in ReNcell CX® cells,different markers of early neural progenitor cells were tested (Table42). Gene expression levels of Nestin and Sox2 were not influenced byASO Seq. ID No. 218b. GFAP mRNA was slightly upregulated after gymnotictransfer with 10 μM ASO Seq. ID No. 218b and in contrast, DCX wasclearly induced after gymnotic uptake of ASO Seq. ID No. 218b.Expression of all tested markers was strongly reduced after TGF-β1treatment (8d) (Table 42, FIG. 19).

TABLE 42 mRNA expression of Nestin, Sox2, GFAP and DCX 8 days aftergymnotic transfer of Seq. ID No. 218b in ReNcell CX ® cells. mRNAexpression levels were determined relative to housekeeping gene GNB2L1using quantitative real-time RT-PCR and then normalized to untreatedcontrol. Cell line ReNcell CX mRNA levels 8 days after gymnotic transferor TGF-β1 exposure Target Nestin Sox2 GFAP DCX n = 4 n = 4 n = 4 n = 4 A1.00 ± 0.18 1.00 ± 0.25 1.00 ± 0.22 1.00 ± 0.32 B 2.5 0.97 ± 0.32 0.88 ±0.33 0.78 ± 0.13 1.31 ± 0.42 μM B 10 0.89 ± 0.16 0.79 ± 0.13 1.02 ± 0.201.44 ± 0.48 μM C 2.5 1.09 ± 0.21 0.93 ± 0.09 0.99 ± 0.14 1.67 ± 0.46 μMC 10 0.90 ± 0.09 0.89 ± 0.11 1.21 ± 0.11 1.95 ± 0.37 μM E 10 0.48 ± 0.120.32 ± 0.06 0.41# ± 0.13  0.05+# ± 0.01   ng/ml A = untreated control, B= Ref. 1, C = Seq. ID No. 218b, E = TGF-β1, ± = SEM, +p < 0.05 inreference to C 2.5 μM, #p < 0.05 in reference to C 10 μM. Statistics wascalculated using the Ordinary-one-way-ANOVA followed by “Tukey's”multiple post hoc comparison.

Conclusion:

Results for mRNA analysis indicate that ASO Seq. ID No. 218b guidesReNcell CX® cells into the direction of an even more stem cell likestate (GFAP upregulation). In addition, induction of DCX indicates anelevated neurogenesis. TGF-β1 treatment results in an oppositedirection.

12.1.3 Results of Antisense-Oligonucleotide Effects on Proliferation ofHuman Neuronal Stem Cells

Further analysis was performed to investigate whether gymnotic transferof ASO Seq. ID No. 218b has really effects on proliferation rate bycounting cells 9 days after repeated gymnotic transfer (3×72 h) anddetermination of Ki67 protein levels 8 days after gymnotic uptake (2×96h).

Results

Cell number was increased after gymnotic uptake of ASO Seq. ID No. 218bin accordance to an increased protein expression of proliferation markerKi67 observed in immunochemical staining of cells (Table 43, FIG. 20).Fluorescence analysis of immunocytochemical staining also revealed aproliferation stop mediated by TGF-β 1.

TABLE 43 Increased cell number 9 days after repeated gymnotic transfer(3 × 72 h) of ReNcell CX ® cells. Cell number was determined using LunaFL ™ Automated Cell Counter Fluorescence and Bright Field (Biozym,#872040) according to manufacturer's instructions. Cell line ReNcell CXCell number alive cells × 10⁵, n = 2 dead cells × 10⁵, n = 2 A 3.34 ±0.09 0.51 ± 0.05 B 2.5 μM 4.34 ± 0.56 0.60 ± 0.09 B 10 μM 4.36 ± 0.960.58 ± 0.09 C 2.5 μM 4.63 ± 1.28 0.47 ± 0.02 C 10 μM 5.24 ± 0.42 0.37 ±0.02 A = untreated control, B = Ref. 1, C = Seq. ID No. 218b, ± = SEM.

Conclusion

Gymnotic transfer of ASO Seq. ID No. 218b in ReNcell CX® cells resultsin an increased cell number, paralleled by an enhanced Ki67 proteinexpression, altogether indicating increased neuronal precursorproliferation.

12.1.3 Results of Antisense-Oligonucleotide Effects on DifferentiationAbility of Human Neuronal Stem Cells

To exclude an influence of ASO Seq. ID No. 218b on cell ability todifferentiate, ASO Seq. ID No. 218b was transferred to cells by gymnoticuptake for 96 h under proliferative conditions (+EGF/FGF). Afterincubation time, medium was changed and to one part of cellsproliferative medium was added whereas to the other part of cellsdifferentiating medium (−EGF/FGF) was added. Afterwards, anothergymnotic transfer for 96 h was performed. Cells were analyzed byexpression levels of neuronal markers Neurofilament N (NeuN) and111-Tubulin.

Results

Immunochemical staining against NeuN (FIG. 23A) and βIII-Tubulin (FIG.23B) demonstrates no effects on the ability to differentiate aftergymnotic ASO transfer under proliferative conditions followed bygymnotic transfer under differentiating conditions. Signal forβIII-Tubulin, a human neuron specific protein, was not influenced by ASOSeq. ID No. 218b under differentiating conditions and was comparable tountreated control. Also NeuN expression was not influenced aftergymnotic transfer under differentiating conditions. Thus, cells arestill capable to differentiate into neural cells. Strikingly, ReNcellCX® cells expressed neuronal marker NeuN and βIII-Tubulin after gymnotictransfer of ASO under proliferative conditions (2×96 h) for bothperiods, indicating that gymnotic transfer of ASO could promote aspecific shift into differentiation of neurons even under proliferativeconditions. In addition, elevated proliferation rates of neuralprecursor cells were observed (Table 43, FIG. 20). Further, stainingagainst NeuN revealed that cells treated with ASO Seq. ID 218b look moreviable compared to all other treatments (FIG. 21A). Obviously, cellswhich were treated with TGF-β1 were significantly less proliferative.

Conclusion

The ability to differentiate was not influenced by inventive ASO Seq. IDNo. 218b. Interestingly, ReNcell CX® cells showed differentiation toneurons after gymnotic transfer under proliferative and differentiatingconditions. This indicates in context to the observation of an increasedproliferation rate, that inventive ASO Seq. ID No. 218b promotesneurogenesis with a tendency towards elevated neuronal differentiation.

12.1.4 Results of Inventive Antisense-Oligonucleotides on Proliferationof Human Neuronal Stem Cells after TGF-β1 Pre-Incubation

To analyze whether gymnotic transfer of ASO Seq. ID No. 218b isefficient in reversing TGF-β1 mediated effects on ReNcell CX® cells,further studies were performed with TGF-β1 pre-incubation for 7 daysfollowed by gymnotic transfer for 8 days (2×96 h).

Results

Gene expression of GFAP (Table 44, FIG. 22A) as an early neuronalmarker, Ki67 (Table 44, FIG. 22B), as a marker for proliferation, andDCX (Table 44, FIG. 22C) as marker for neurogenesis were elevated aftersingle ASO treatment, whereas TGF-β1 resulted in the opposite. Inaddition, 7 days after TGF-β1 pre-incubation, inventive ASO treatmentreversed TGF-β1-induced effects. Thus the analysis demonstrates that ASOSeq. ID No. 218b is potent in recovering TGF-β1 mediated effects uponstem cell and proliferation markers

TABLE 44 mRNA expression of GFAP, Ki67 and DCX 7 days after TGF- β1pre-incubation followed by 2 × 96 h gymnotic transfer of Seq. ID No.218b in ReNcell CX ® cells. mRNA expression levels were determinedrelative to housekeeping gene GNB2L1 using quantitative real-time RT-PCRand then normalized to untreated control. Cell line ReNcell CX mRNAlevels 7 d after TGF-β1 pre-incubation followed by 2 × 96 h gymnotictransfer Target GFAP Ki67 DCX n = 2 n = 1 n = 2 A 1.00 ± 0.20 1.00 1.00± 0.16 B 10 μM 1.62 ± 0.15 0.91 1.52 ± 0.24 C 10 μM 2.23 ± 0.52 1.524.82 ± 1.15 E 10 ng/ml 0.76 ± 0.01 0.48 0.68 ± 0.03 E 10 ng/ml + B 10 μM0.58 ± 0.07 0.61 0.83 ± 0.10 E 10 ng/ml + C 10 μM 2.04 ± 1.04 7.40 1.55± 0.24 A = untreated control, B = Ref. 1, C = Seq. ID No. 218b, E =TGF-β1, ± = SEM, Statistics was calculated using theOrdinary-one-way-ANOVA followed by “Tukey's” multiple post hoccomparisons.

Conclusion

Results indicate that adult neurogenesis could be reactivated byinventive TGF-R_(II) specific ASO-mediated blocking of TGF-β signaling.

Taken together, TGF-R_(II) specific ASO Seq. ID No. 218b rescued cellsfrom TGF-β mediated stem cell quiescence and promotes adult neurogenesiswithout having an impact on differentiation. This makes it an idealtreatment drug for brain repair.

Example 13: Determination of Therapeutic Activity of InventiveAntisense-Oligonucleotides Disease Progression of ALS in SOD1 Mice

To analyze the therapeutic potential of ASOs as a medication foramyotrophic lateral sclerosis (ALS) male and female transgenic, SOD1G93A mice were treated with different doses of inventive ASOs by icyadministration into the lateral ventricle via osmotic ALZET® minipumps.In addition, riluzole was used as a reference. Riluzole is a drug usedto treat amyotrophic lateral sclerosis and is marketed by SanofiPharmaceuticals. It delays the onset of ventilator-dependence ortracheostomy in selected patients and may increase survival byapproximately two to three months

Description of Method:

For long-lasting central infusion an icy cannula attached to an Alzet®osmotic minipump (infusion rate: 0.25 μl/h, Alzet®, Model 2004,Cupertino, USA), was stereotaxically implanted under isofluraneanesthesia (Baxter, GmbH, Germany) and semi-sterile conditions. Eachosmotic minipump was implanted subcutaneously in the abdominal regionvia a 1 cm long skin incision at the neck of the mouse and connectedwith the icy cannula by silicone tubing. Animals were placed into astereotaxic frame, and the icy cannula (23G, 3 mm length) was loweredinto the right lateral ventricle (posterior 0.3 mm, lateral 1 mm, depth3 mm relative to bregma). The cannula was fixed with two stainless steelscrews using dental cement (Kallocryl, Speiko® Dr. Speier GmbH, Münster,Germany). The skin of the neck was closed with sutures. During surgery,the body temperature was maintained by a heating pad. To avoidpost-surgical infections, mice were locally treated with Betaisodona®(Mundipharma GmbH, Limburg, Germany) and received 0.1 ml antibiotics(sc, Baytril® 2.5% Bayer Vital GmbH, Leverkusen, Germany). The tubingwas filled with the respective solution. To determine the effects ofASOs on the development and the progression of ALS, the onset ofsymptoms, paresis, and survival were used as in vivo endpoints. At theage of nine weeks, mice were sacrificed and brains were removed forneuropathology analysis. Histological verification of the icyimplantation sites was performed at 40-pm coronal, cresyl violet-stainedbrain sections.

The inventive ASOs exert potential effects in in vitro experiments.Quite in line, the rodent cross-reactive inventive ASOs with Seq. ID No.143aj, Seq. ID No. 143h and Seq. ID No. 210q were also effective in theabove experiments proving an effect in the treatment of ALS modelanimals. The ASOs of the present invention demonstrating nocross-reactivity exert more potential effects in in vitro experiments.As a result, it is assumed that these inventive ASOs are also moreeffective in in vivo set ups for non-human primates and humans andtherefore act as a highly potent medication for preventing or treatingTGF-β1 induced inhibition of neural stem and progenitor proliferation,and thereby treating ALS and other neurodegenerative disorders.

Examples 14: Determination of the Therapeutic Activity ofAntisense-Inventive ASOs Directed to TGF-R_(II) on Disease Developmentand Progression of Huntington's Disease in R6/2 Mice

To analyze the therapeutic potential of ASOs as a medication forHuntington's disease (HD), male and female transgenic R6/2 mice weretreated with different doses of inventive TGF-R_(II) specific ASO by icyadministration into the lateral ventricle via osmotic minipumps.

Description of Method:

For chronic central infusion, mice underwent surgery for an icy cannulaattached to an Alzet® osmotic minipump (infusion rate: 0.25 μl/h,Alzet®, Model 2004, Cupertino, USA) at the age of five weeks. Thecannula and the pump were stereotaxically implanted underketamine/xylacin anesthesia (Baxter, GmbH, Germany) and semi-sterileconditions. Each osmotic minipump was implanted subcutaneously in theabdominal region via a 1 cm long skin incision at the neck of the mouseand connected with the icy cannula by a silicone tubing. Animals wereplaced into a stereotaxic frame, and the icy cannula (23G, 3 mm length)was lowered into the right lateral ventricle (posterior 0.3 mm, lateral1 mm, depth 3 mm relative to bregma). The cannula was fixed with twostainless steel screws using dental cement (Kallocryl, Speiko®-Dr.Speier GmbH, Münster, Germany). The skin of the neck was closed withsutures. During surgery, the body temperature was maintained by aheating pad. To avoid post-surgical infections, mice were locallytreated with Betaisodona® (Mundipharma GmbH, Limburg, Germany) andreceived 0.1 ml antibiotics (sc, Baytril® 2.5% Bayer Vital GmbH,Leverkusen, Germany). The tubing was filled with the respectivesolution. To determine the effects of ASOs on the development and theprogression of HD the onset of symptoms, grip strength, general motoric,and survival were used as in vivo endpoints. At the age of nine weeks,mice were sacrificed and brains were removed for histologicalanalyzation. Histological verification of the icy implantation sites wasperformed at 40-pm coronal, cresyl violet-stained brain sections.

The inventive ASOs exert potential effects in in vitro experiments.Quite in line, the rodent cross-reactive inventive ASOs with Seq. ID No.143aj, Seq. ID No. 143h and Seq. ID No. 210q were also effective in theabove experiments proving an effect in the treatment of Huntington modelanimals. The ASOs of the present invention demonstrating nocross-reactivity exert more potential effects in in vitro experiments.As a result, it is assumed that these inventive ASOs are also moreeffective in in vivo set ups for non-human primates and humans andtherefore act as a highly potent medication for preventing or treatingTGF-β1 induced inhibition of neural stem and progenitor proliferation,and thereby treating HD and other neurodegenerative disorders.

Example 15: Determination of Therapeutic Activity of the Inventive ASOson Disease Progression of TGFβ-Induced Hydrocephalus and AssociatedCognitive Deficits in Fischer-344 Rats

The goal of the present study is to treat animals suffering from theTGFβ induced effects on i) neural stem cell proliferation andneurogenesis, ii) formation of hydrocephalus, and iii) spatial learningdeficits by intraventricular infusion of inventive ASO in adose-dependent manner.

Description of Method:

Osmotic minipumps for intracerebroventricular infusion were implantedinto female Fischer-344 rats of 180 to 200 g body weight (n_(total)=70,n_(group)=10). Infused were a) artificial cerebrospinal fluid (aCSF:148.0 mM NaCl, 3.0 mM KCl, 1.4 mM CaCl₂), 0.8 mM MgCl₂, 1.5 mM Na₂HPO₄,0.2 mM NaH₂PO₄, 100 μg/ml rat serum albumin, 50 μg/ml Gentamycin, pH7.4) as control, or b) TGF-β1 1 μg/mL in aCSF using an Alzet® osmoticpump 2004 with flow rate of 0.25 μl/h for 14 days. After 14 days thepumps are changed and Alzet® osmotic pumps 2004 (flow rate 0.25 μl/h)were used for the following infusions: aCSF or TGF-β1 (1 μg/ml) incombination with varying concentrations of TGF-R_(II) ASO (1.1 mmol/L,3.28 mmol/l, 9.84 mmol/I) or scrambled ASO (3.28 mmol/I) were infused(2×4 weeks). During the last four days of the infusion period, animalsreceived a daily intraperitoneal injection of BrdU (50 mg/kg of bodyweight) to label proliferating cells. Pumps are removed, and two weekslater animals are functionally analyzed in a spatial learning test(Morris-Water-Maze) for 14 days. One day later, animals are perfusedwith 0.9% NaCl, brains are removed, the ipsilateral hemisphere ispostfixed in 4% paraformaldehyde for quantitative histological analysisof PCNA, BrdU, DCX, BrdU/NeuN, and BrdU/GFAP, and for stereologicalanalysis of the volume of the lateral ventricles as a measure for thehydrocephalus. The contralateral hemisphere is further dissected anddifferent areas (ventricle wall, hippocampus, cortex) are processed forquantitative RT-PCR to analyze TGF-R_(II) expression levels. MR imageswere taken of 4 animals of group 1, group 3, and group 6 at day fourbefore pump implantation, one week after pump implantation, at the dayof the first pump change and from then on every 2 weeks until the end ofthe infusion period. Histological verification of the icy implantationsites was performed at 40-pm coronal, cresyl violet-stained brainsections.

TABLE 46 Treatment scheme and the group classification of theHydrocephalus experiment. Group 2. aCSF + 4. TGF-β1 + 5.-7. TGF-β1 + 1.aCSF ASO 3. TGF-β1 scramb-ASO ASO treatment aCSF- aCSF plus TGF-β1-TGF-β1 plus TGF-β1 plus infusion ASO infusion infusion ASO infusion ASOinfusion treatment week 1 week 1 and 2: week 1 and 2: week 1 and 2: week1 and 2: scheme to 10 aCSF 1 μg/ml TGF-β1: 1 μg/ml TGF-β1: 1 μg/ml week3 to 10: week 3 to 10: week 3 to 10: week 3 to 10: ASO: 1 μg/ml TGF-β1:1 μg/ml TGF-β1: 1 μg/ml 3.28 mmol/l scramb.-ASO: ASO: 1.1 mmol/l 3.28mmol/l 3.28 mmol/l 9.84 mmol/l n 10 10 10 10 10 per dose n-total 10 1010 10 30

The inventive ASOs exert potential effects in in vitro experiments.Quite in line, the rodent cross-reactive inventive ASOs with Seq. ID No.143aj, Seq. ID No. 143h and Seq. ID No. 210q were also effective in theabove experiments proving an effect in the treatment of Hydrocephalusmodel animals. The ASOs of the present invention demonstrating nocross-reactivity exert more potential effects in in vitro experiments.As a result, it is assumed that these inventive ASOs are also moreeffective in in vivo set ups for non-human primates and humans andtherefore act as a highly potent medication for preventing or treatingTGF-β1 induced inhibition of neural stem and progenitor proliferation,and thereby treating Hydrocephalus and other neurodegenerativedisorders.

Example 16: Determination of Therapeutic Activity of theAntisense-Oligonucleotides Directed to TGF-R_(II) on Rehabilitation ofSpinal Cord Injury in Fischer 344 Rats

To analyze the therapeutic potential of ASOs as a medication for spinalcord injury (SCI), male and female Fischer-344 rats were treated withdifferent doses of inventive ASOs by icy administration into the lateralventricle via osmotic minipumps.

Description of Method:

SCI was simulated by cervical tungsten wire knife dorsal columntransection at the C3 level. In the next step, for chronic centralinfusion rats, (180-200 g body weight) underwent surgery for an icycannula attached to an Alzet® osmotic minipump (infusion rate: 0.25μl/h, Alzet®, Model 2004, Cupertino, USA). The cannula and the pump werestereotaxically implanted under ketamine/xylacin anesthesia (Baxter,GmbH, Germany) and semi-sterile conditions. Each osmotic minipump wasimplanted subcutaneously in the abdominal region via a 1 cm long skinincision at the neck of the rat and connected with the icy cannula by asilicone tubing. Animals were placed into a stereotaxic frame, and theicy cannula (23G, 3 mm length) was lowered into the right lateralventricle (posterior 1.0 mm, lateral 1.0 mm, depth 1.8 mm relative tobregma). The cannula was fixed with two stainless steel screws usingdental cement (Kallocryl, Speiko®-Dr. Speier GmbH, Münster, Germany).The skin of the neck was closed with sutures. During surgery, the bodytemperature was maintained by a heating pad. To avoid post-surgicalinfections, rats were locally treated with Betaisodona® (MundipharmaGmbH, Limburg, Germany) and received 0.5 ml antibiotics (sc, Baytril®2.5% Bayer Vital GmbH, Leverkusen, Germany). The tubing was filled withthe respective solution. To determine the effects of ASOs on therehabilitation process following SCI, 4 weeks post-surgery an in vivoMRI structural analysis was performed (3T MRI, Allegra Siemens, phasedarray—small animal coil). 6 weeks after surgery, animals were sacrificedand the spinal cord was removed for histological and immunohistochemicalanalysis. Histological verification of the icy implantation sites wasperformed at 40-pm coronal, cresyl violet-stained brain sections.

The inventive ASOs exert potential effects in in vitro experiments.Quite in line, the rodent cross-reactive inventive ASOs with Seq. ID No.143aj, Seq. ID No. 143h and Seq. ID No. 210q were also effective in theabove experiments proving an effect in the treatment of aFischer-344—rat spinal cord paraplegia model. In MRI images andneuropathological analysis, the inventive ASOs showed high treatmentefficacy. The ASOs of the present invention demonstrating nocross-reactivity exert more potential effects in in vitro experiments.As a result, it is assumed that these inventive ASOs are also moreeffective in in vivo set ups for non-human primates and humans andtherefore act as a highly potent medication for preventing or treatingTGF-β1 induced inhibition of neural stem and progenitor proliferation,and thereby treating spinal cord injury and other neurodegenerativedisorders.

Example 17: ASO-Mediated Effects on Proliferation of Human Lung CancerCell Line A549

mRNA of Ki67, p53, Caspase 8 (Casp8) and of DNA-binding proteininhibitor 2 (ID2) were analyzed as representative markers onproliferation in several tumor cells. It is known from previous studies,that expression of tumor suppressor gene p53 and ID2 is oftendramatically elevated in tumor tissues. Ki67 is a proliferation markerand Casp8 is an indicator for apoptosis. In addition, cell numbers weredetermined after gymnotic transfer.

Description of Method:

A549 were cultured as described above. For treating cells, medium wasremoved and replaced by fresh full medium in 24-well culture dishes(Sarstedt #83.1836.300) (30,000 cells/well), 6-well culture dishes(Sarstedt #83.3920.300) (50,000 cells/well) or 8-x-well cell cultureslide dishes (Sarstedt #94.6140.802) (20,000 cells/well) (0.5 ml for24-well and 8-well cell culture slide dishes and 1 ml for 6-well dishes)and were incubated overnight at 37° C. and 5% CO₂. To analyze mRNAexpression and influence on proliferation, cells were treated with Ref.1(Scrambled control) and ASO Seq. ID No. 218b at concentrations of 2.5 μMand 10 μM and were incubated for 72 h at 37° C. and 5% CO₂. Treatmentincluding medium replacement was repeated for 3 times every 72 h (12days in total). For immunocytochemical analysis of proliferation (Ki67),gymnotic transfer of ASO Seq. ID No. 218b was limited to 72 h.Afterwards, cells were washed twice with PBS and subsequently used forprotein isolation (6-well dishes), immunocytochemistry (in 8-well cellculture slide dishes), proliferation curve and RNA isolation (24-welldishes). Protocols for RNA, protein and immunocytochemistry wereperformed as described above. For proliferation curve, remaining cellswere harvested from 24-well dishes for determination of cell number. Forthis purpose, remaining cells were washed with PBS (2×), treated withaccutase (500 μl/well) and incubated for 7 min at 37° C. Afterwards 500μl medium was added and cell number was determined using Luna FL™Automated Cell Counter Fluorescence and Bright Field (Biozym, #872040)according to manufacturer's instructions. Briefly, 18 μl of the cellsuspension was added to 2 μl of acridine orange/propidium iodide assayviability kit (Biozym #872045). After 1 min of settling, 10 μl wereadded onto Cell Counting Slide (Biozym #872011). Cells were counted andcalculated in distinction of alive and dead cells.

17.1 Results for ASO Seq. ID No. 218b

mRNA analysis showed reduced Ki67, p53 and ID2 expression levels 12 daysafter gymnotic transfer of ASO Seq. ID No. 218b. In contrast, Casp8 waselevated at low levels of ASO Seq. ID No. 218b (Table 46). Theseobservations indicate that a reduced tumor growth is associated with aslight increase in apoptotic cells. Furthermore, Western Blot analysisshowed reduction in protein level of Ki67 and pAkt 12 days aftergymnotic transfer of inventive ASOs (Table 47). Immunochemicalexamination of A549 cells after gymnotic transfer of ASO Seq. ID No.218b showed a reduced level of Ki67 signals in comparison to scrambledcontrol for both concentrations applied (FIG. 23). Finally, cell numberof A549 cells was reduced about nearly 50% 12 days after gymnotictransfer of ASO Seq. ID No. 218b (Table 48).

TABLE 46 mRNA expression of Ki67, p53, Casp8 and ID2, 12 days aftergymnotic transfer of ASO Seq. ID No. 218b in A549 cells. Regulation ofexamined genes demonstrates diminished proliferation rates aftergymnotic transfer of inventive ASOs. Reduced ID2 mRNA levels arebeneficial in dampening expansion of tumor cells. mRNA expression levelswere determined relative to housekeeping gene GNB2L1 using quantitativereal-time RT-PCR normalized to untreated control. Cell line A549 mRNAlevels 12 days after repeated gymnotic transfer (4 × 72 h) Target Ki67p53 Casp8 ID2 n = 2 n = 2 n = 2 n = 2 A 1.00 ± 0.37 1.00 ± 0.31 1.00 ±0.05 1.00 ± 0.03 B 2.5 μM 0.92 ± 0.05 1.06 ± 0.02 1.36 ± 0.37 0.73 ±0.01 B 10 μM 0.96 ± 0.03 1.11 ± 0.92 1.52 ± 0.15 0.82 ± 0.15 C 2.5 μM0.55 ± 0.33 0.27 ± 0.04 1.59 ± 0.48 0.59 ± 0.01 C 10 μM 0.57 ± 0.20 0.53± 0.07 0.98 ± 0.17 0.35 ± 0.02 A = untreated control, B = Ref. 1, C =Seq. ID No. 218b, ± = SEM, Statistics was calculated using theOrdinary-one-way-ANOVA followed by “Tukey{acute over ( )}s” multiplepost hoc comparisons.

TABLE 47 Densitometric analysis of Ki67 and pAkt Western Blot.Downregulation of Ki67 and pAkt protein 12 days after gymnotic transferwith TGF-R_(II) specific ASO Seq. ID No. 218b was observed in A549cells. Protein levels were determined relative to housekeeping geneGAPDH using Image Studio ™ Lite Software and were then normalized tountreated control. Cell line A549 protein levels 12 days after repeatedgymnotic transfer (4 × 72 h) Target Ki67 pAKT n = 1 n = 1 A 1.00 1.00 B10 μM 1.18 0.80 C 10 μM 0.57 0.39 A = untreated control, B = Ref. 1, C =Seq. ID No. 218b, ± = SEM.

TABLE 48 Cell numbers 12 days after repeated gymnotic transfer. Cellnumbers were determined 12 days after repeated gymnotic transfers (4 ×72 h) of A549 cells using Luna FL ™ Automated Cell Counter Fluorescenceand Bright Field (Biozym, #872040) according to manufacturer'sinstructions. Cell line A549 cell number 12 days after repeated gymnotictransfer (4 × 72 h) Cell number alive cells × 10⁵ dead cells × 10⁵ n = 3n = 3 A 4.25 ± 0.50 0.47 ± 0.09 B 10 μM 3.88 ± 0.95 0.31 ± 0.11 C 10 μM2.35 ± 0.07 0.35 ± 0.16 A = untreated control, C = Seq. ID No. 218b, ± =SEM.

Conclusion

These observations indicate that reduced tumorous growth is associatedwith an increase in apoptotic cells. Notably, ID2, which is a possibletherapeutic target gene in tumors, is reduced after gymnotic transfer ofTGF-R_(II) specific ASO Seq. ID No. 218b.

Taken together, ASO Seq. ID No. 218b is efficient in minimizingproliferation rates and reduces tumor promoting gene expression.

Example 18: Effect of ASO Gymnotic Transfer on Proliferation of SeveralTumor Cell Lines

TGF-β signaling is a critical pathway in cancer development. On the onehand TGF-β promotes factors, which act tumor suppressive but on theother hand, this growth factor leads to stimulation of cell migration,cell invasion, cell proliferation, immune regulation, and promotes anenvironmental reorganization in advantage to progression and metastasisof tumor cells. Thus, TGF-β is a key target in cancer treatment. mRNAand protein levels of proliferation marker (Ki67) and cell numbers weredetermined after gymnotic uptake of inventive ASOs as markers ofproliferation rate in tumor cells. Furthermore, mRNA levels of tumorsuppressor gene p53 and of DNA-binding protein inhibitor 2 (ID2) wereexamined.

Description of Methods

Several tumor cell lines were cultured as described above (Table 10).For treating cells, medium was removed and replaced by fresh full mediumin 24-well culture dishes (Sarstedt #83.1836.300) (30,000 cells/well),6-well culture dishes (Sarstedt #83.3920.300) (50,000 cells/well) (0.5ml for 24-well and 1 ml for 6-well dishes) and were incubated overnightat 37° C. and 5% CO₂. To analyze mRNA expression and influence onproliferation, cells were treated with Ref.1 (Scrambled control) and ASOSeq. ID No. 218b at concentrations of 2.5 μM and 10 μM and wereincubated for 72 h at 37° C. and 5% CO₂. Treatment including mediumreplacement was repeated 3 times every 72 h (12 days in total). Forharvesting, cells were washed twice with PBS and subsequently used forRNA isolation (24-well dishes), protein isolation (6-well dishes), orproliferation curve. Protocols for RNA and protein isolation wereperformed as described above. Before counting cells for proliferationcurve, cells were analyzed by using light microscopy (Nikon, TS-100 FLED #MFA33500). Remaining cells were then harvested from 24-well dishesfor determination of cell number. For this purpose, remaining cells werewashed with PBS (2×), treated with accutase (500 μl/well) and incubatedfor 5-7 min at 37° C. Afterwards 500 μl medium was added and cell numberwas determined using Luna FL™ Automated Cell Counter Fluorescence andBright Field (Biozym, #872040) according to manufacturer's instructions.Briefly, 18 μl of the cell suspension were added to 2 μl of acridineorange/propidium iodide assay viability kit (Biozym #872045). After 1min of settling, 10 μl were added onto Cell Counting Slide (Biozym#872011). Cells were counted and calculated in distinction of alive anddead cells.

18.1 Results for Seq. ID No. 218b

Ki67 mRNA levels were efficiently decreased independently (A549, L3.6pl,Panc-1) or dependently (HT-29, Panc-1, CaCo2) of used ASOconcentrations, 12 days after gymnotic transfer (Table 40). Geneexpression level of p53 was also affected in A549, HT-29, K562, KG-1,CaCo2 and TMK-1 by tested ASO (Table 50). Verification of reduced Ki67protein expression was shown for A549, L3.6pl, TMK-1, HT-29 and K562(Table 51). Notably, ID2 mRNA expression showed a consistent efficientlyand dose-dependently downregulation in A549, HT-29, K562 and TMK-1 cellsmediated by ASO Seq. ID No. 218b (Table 51). In addition, ASO Seq. IDNo. 218b resulted in a reduced proliferation rate of several tumor celllines (Table 53). A dose-dependent decrease of cell number wasrecognized for HPAFII, MCF-7, KG1, K562, U937 and HTZ-19 cells. Lungcancer cells (A549) showed approx. 50% reduction of cell numberselicited by ASO Seq. ID No. 218b. Reduced cell numbers were additionallyconfirmed by light microscopy for HPAFII, K562, MCF-7, Panc-1 and HTZ-1,12 days after gymnotic transfer of ASO Seq. ID No. 218b (FIG. 24).

TABLE 49 mRNA expression of proliferation marker Ki67. 12 days aftergymnotic transfer of ASO Seq. ID No. 218b in A549, HT-29, L3.6pl, KG1,Panc-1 and CaCo2 cells, Ki67 mRNA was decreased in all cell lines,respectively. mRNA levels were determined relative to housekeeping geneGNB2L1 using quantitative real-time RT-PCR normalized to untreatedcontrol. Target Ki67 mRNA levels 12 days after repeated gymnotictransfer (4 × 72 h) Cell line A549 HT-29 L3.6pl KG1 Panc-1 CaCo2 n = 2 n= 2 n = 2 n = 1 n = 1 n = 1 A 1.00 ± 1.00 ± 1.00 ± 1.00 1.00 1.00 0.370.00 0.25 B 2.5 μM 0.92 ± 0.89 ± 0.93 ± 0.72 0.76 1.21 0.05 0.46 0.03 B10 μM 0.96 ± 0.60 ± 0.96 ± 0.76 0.79 1.07 0.03 0.11 0.16 C 2.5 μM 0.55 ±0.34 ± 0.42 ± 0.16 0.68 0.99 0.33 0.11 0.03 C 10 μM 0.57 ± 0.17 ± 0.64 ±0.33 0.37 0.37 0.20 0.02 0.05 A = untreated control, B = Ref. 1, C =Seq. ID No. 218b, ± = SEM, Statistics was calculated using theOrdinary-one-way-ANOVA followed by “Tukey's” multiple post hoccomparisons.

TABLE 50 mRNA expression of tumor suppressor p53. 12 days after gymnotictransfer of ASO Seq. ID No. 218b in A549, HT-29, K562, KG1, CaCo2 andTMK-1 cells, p53 mRNA was decreased in all cell lines, respectively.mRNA expression levels were determined relative to housekeeping geneGNB2L1 using quantitative real-time RT- PCR and then normalized tountreated control. Target p53 mRNA levels 12 days after repeatedgymnotic transfer (4 × 72 h) Cell line A549 HT-29 K562 KG1 TMK-1 CaCo2 n= 2 n = 1 n = 1 n = 1 n = 1 n = 1 A 1.00 ± 1.00 1.00 1.00 1.00 ± 1.000.31 0.04 B 2.5 μM 1.06 ± 0.72 0.90 1.37 0.74 ± 0.82 0.02 0.11 B 10 μM1.11 ± 0.68 1.35 0.87 0.71 ± 1.25 0.92 0.15 C 2.5 μM 0.27 ± 0.51 0.270.65 0.14* ± 0.99 0.04 0.14 C 10 μM 0.53 ± 0.32 0.46 0.67 0.21* ± 0.300.07 0.05 A = untreated control, B = Ref. 1, C = Seq. ID No. 218b, ± =SEM, *p < 0.05 in reference to A, Statistics was calculated using theOrdinary-one-way-ANOVA followed by “Tukey's” multiple post hoccomparisons.

TABLE 51 mRNA expression of ID2. 12 days after gymnotic transfer of ASOSeq. ID No. 218b in A549, HT-29, K562 and TMK-1 cells, ID2 mRNA wasdose-dependently downregulated in all cell lines, respectively. mRNAexpression levels were determined relative to housekeeping gene GNB2L1using quantitative real-time RT-PCR and then normalized to untreatedcontrol. Target ID2 mRNA levels 12 days after repeated gymnotic transfer(4 × 72 h) Cell line A549 HT-29 K562 TMK-1 n = 2 n = 1 n = 1 n = 1 A1.00 ± 0.03 1.00 1.00 ± 0.23 1.00 ± 0.23 B 2.5 μM 0.73 ± 0.01 0.93 0.97± 0.15 0.88 ± 0.15 B 10 μM 0.82 ± 0.15 1.00 0.82 ± 0.05 0.82 ± 0.05 C2.5 μM 0.59 ± 0.01 0.31 0.70 ± 0.10 0.70 ± 0.10 C 10 μM 0.35 ± 0.02 0.250.29* ± 0.09  0.30* ± 0.09  A = untreated control, B = Ref. 1, C = Seq.ID No. 218b, ± = SEM, *p < 0.05 in reference to A, Statistics wascalculated using the Ordinary-one-way-ANOVA followed by “Tukey{acuteover ( )}s” post hoc multiple comparisons.

TABLE 52 Densitometric analysis of Ki67 Western Blot. Downregulation ofKi67 protein after gymnotic transfer with ASO Seq. ID No. 218b wasrecognized. Protein level was quantified relative to housekeeping genealpha-tubulin using Image Studio ™ Lite Software and normalized tountreated controls. Target Ki67 protein level 12 days after repeatedgymnotic transfer (4 × 72 h) Cell line A549 L3.6pl TMK-1 HT29 K562 n = 1n = 2 n = 2 n = 2 n = 1 A 1.00 1.00 ± 0.00 1.00 ± 0.00 1.00 ± 0.00 1.00B 10 μM 1.18 0.59 ± 0.00 0.75 ± 0.00 1.19 ± 0.68 1.05 C 10 μM 0.57 0.19± 0.17 0.53 ± 0.26 0.69 ± 0.05 0.35 A = untreated control, B = Ref. 1, C= Seq. ID No. 218b. Statistics was calculated using theOrdinary-one-way-ANOVA followed by “Tukey{acute over ( )}s” post hoccomparisons.

TABLE 53 Cell numbers in several cancer cell lines 12 days afterrepeated gymnotic transfer (4 × 72 h). ASO Seq. ID No. 218b wastransferred to several cancer cell lines. Cell numbers were determinedusing Luna FL ™ Automated Cell Counter Fluorescence and Bright Field(Biozym, #872040) according to manufacturer's instructions. Treatment AB 2.5 μM B 10 μM C 2.5 μM C 10 μM cell number × 10⁵ Cell Line a d a d ad a d a d n p = A549 4.25 ± 0.47 ± 3.88 ± 0.31 ± 2.35 ± 0.35 ± 3 0.500.09 0.95 0.11 0.07 0.16 HPAFII 2.80 ± 0.35 ± 2.88 ± 0.36 ± 2.56 ± 0.39± 0.66 ± 0.25 ± 0.20 ± 0.06 ± 2 0.33 0.11 2.04 0.06 0.45 0.06 0.47 0.070.09 0.02 KG1 17.40 ± 0.43 ± 16.5 ± 0.58 ± 13.80 ± 0.26 ± 10.90 ± 0.59 ±7.63 ± 0.48*+ ± 3 A vs. C 10 μM *p < 0.01 3.00 0.16 0.85 0.24 0.80 0.170.20 0.18 3.08 0.14 B 2.5 μM vs. C 10 μM +p < 0.01 C 10 μM vs. D 10 μM#p < 0.01 K562 10.93 ± 1.37 ± 7.44 ± 2.40 ± 6.40 ± 2.36 ± 5.60 ± 2.66 ±3.33 ± 0.62* ± 3 A vs. C 10 μM *p < 0.01 1.58 0.40 1.05 0.62 0.38 0.300.08 0.41 0.54 0.07 MCF-7 6.73 2.37 6.51 1.57 6.51 3.35 5.21 1.64 2.470.73 1 U937 26.43 ± 7.04 ± 14.5 ± 2.88 ± 17.67 ± 2.36 ± 11.34* ± 3.07 ±7.56* ± 2.25 ± 3 A vs. C 2.5 μM *p < 0.01 2.05 0.28 2.73 0.37 0.50 0.302.85 0.97 1.49 0.44 A vs. C 10 μM *p < 0.01 Panc-1 2.16 ± 0.11 ± 1.82 ±0.15 ± 2.98 ± 0.16 ± 1.15* ± 0.07 ± 1.20*+ ± 0.36 ± 3 A vs. C 2.5 μM *p< 0.05 0.08 0.02 0.36 0.04 0.27 0.02 0.51 0.02 0.23 0.02 A vs. C 10 μM*p < 0.05 B 10 μM vs. C 10 μM +p < 0.01 HTZ-19 2.06 ± 3.05 ± 2.57 ± 1.78± 2.55 ± 1.22 ± 1.78 ± 0.88 ± 1.17+ ± 0.49 ± 3 B 10 μM vs. C 10 μM +p <0.02 0.36 0.16 0.15 0.22 0.15 0.25 0.09 0.14 0.05 0.05 A = untreatedcontrol, B = Ref. 1, C = Seq. ID No. 218b, a = alive cells, d = deadcells. ± = SEM. Statistics was calculated using theOrdinary-one-way-ANOVA followed by “Tukey's” post hoc comparisons test.

Conclusion

Modulation of Ki67, p53 and ID2 mRNA by ASO Seq. ID No. 218b indicates abeneficial effect in dampening tumor expansion in several organs andwith different origin. Ki67, ID2 and p53 are known to be upregulated andpromote cell proliferation in different cancer types. Proliferationmarker Ki67, p53 and ID2 were efficiently downregulated. Cell countingand light microscopy of several tumor cells 12 days after gymnotictransfer revealed ASO Seq. ID No. 218b as a potent agent to reduce cellproliferation.

Taken together, TGF-R_(II) specific ASO Seq. ID No. 218b was efficientlyreducing proliferation rates parallel to recognized mRNA modulations ofKi67, p53 and ID2. These data suggest that the inventive ASOs arepromising drug candidates for dampening tumor cell progression andmetastasis of tumor cells.

Example 19: Analysis of the Effect of the Antisense-Oligonucleotides toAngiogenesis in Several Tumor Cell Lines

Modulation of angiogenesis is essential for organ growth and repair. Animbalance in blood vessel growth contributes to different diseases likee.g. tumor growth, ischemia, inflammatory and immune disorders. TGF-β isknown to be a pro-angiogenic factor. This may be most relevant ininflammatory and neoplastic processes, when overshooting angiogenesis isresponsible for disease progression. These effects may go hand in handwith TGF-β1 induced fibrosis. Therefore Inhibition of TGF-β signaling byTGFR_(II) specific ASO may represent an adequate therapeutic approach.

To test this assumption, these ASOs were transferred to several tumorcell lines by gymnotic uptake. 12 days after repeated gymnotictransfers, cell supernatant was analyzed for protein levels ofpro-angiogenic factors by multiplex analysis. This technology allowedinvestigation of multiple pro-angiogenic proteins (VEGF, Tie-2, FLt-1,PIGF and bFGF) by electro-chemiluminescence. Vascular endothelial growthfactor (VEGF) is a potent tumor secreted cytokine that promotesangiogenesis and therewith contributes to e.g. tumor proliferation.Tie-2 is a protein which is expressed from actively growing bloodvessels. Fms-like tyrosine kinase 1 (Flt-1), also known as vascularendothelial growth factor receptor 1 (VEGFR1), is a transmembranetyrosine receptor kinase that is highly expressed in vascularendothelial cells and Placental Growth Factor (PIGF) acts together withVEGF and is upregulated under pathological conditions e.g. in tumorformation. Besides, basic Fibroblast Growth Factor (bFGF) is a growthfactor that also induces angiogenesis. PAI-1 is a target gene of TGF-βand mediates scar formation and angiogenic effects of TGF-β. Therefore,PAI-1 demonstrates also a key factor for tumor invasion and metastasis.Patients showing a high PAI-1 concentration level are considered to apoor prognostic factor e.g. in breast cancer, lung, colorectal andgastric cancer. High PAI-1 concentrations also are a risk factor fordiseases where thrombosis plays a role (e.g. myocardial infarction,stroke). Thus, PAI-1 mRNA regulation by TGF-β specific antisenseoligonucleotides was also tested.

Description of Methods:

Tumor cell lines were cultured as described above (Table 10). Fortreating cells, medium was removed and replaced by fresh full medium in24-well culture dishes (Sarstedt #83.1836.300) (30,000 cells/well)incubated overnight at 37° C. and 5% CO₂. The next day, Ref.1 (Scrambledcontrol) and ASO Seq. ID No. 218b (were added to refreshed medium atconcentrations of 2.5 and 10 μM and were incubated for 72 h at 37° C.and 5% CO₂. Treatment including medium replacement was repeated 3 timesevery 72 h (12 days in total). Afterwards cell supernatant was collectedand analyzed by a MesoScale Discovery® Assay (MSD Discovery). Thistechnology allowed investigation of multiple pro-angiogenic proteins(VEGF, Tie-2, FLt-1, PIGF and bFGF) by electro-chemiluminescence.Experiment performance and information about the individual growthfactors were extracted by manufacturer instructions (MSD MesoScaleDiscovery®, #K15198G). The results were evaluated by GraphPad Prism® 6.0Software.

Afterwards, cells were washed twice with PBS and subsequently used forRNA isolation (24-well dishes) to analyze, whether gymnotic transfer ofASO may regulate mRNA levels of Plasminogen Activator inhibitor-1(PAI-1) by real-time RT-PCR. Protocols and primers were used and listedas described before.

19.1 Results for Seq. ID 218b

Table 54 demonstrates that PAI-1 mRNA was downregulated in adose-dependent manner in several tested cancer cells (A549: lung cancer,HPAFII: pancreatic adenocarcinoma, HT-29: colorectal adenocarcinoma,HTZ-19: melanoma, TMK-1: gastric carcinoma, THP-1: monocytic leukemia)after repeated gymnotic transfer of ASO Seq. ID No. 218b. In addition,VEGF protein levels in stimulated cell supernatants showed also adose-dependent decrease in A549, HTZ-19, HPAFII and PC3M (prostaticadenocarcinoma). For HPAFII and PC3M cells downregulation wassignificant (Table 55). Influence of ASO Seq. ID No. 218b to bFGFconfirmed observations for VEGF, meaning that ASO Seq. ID No. 218b ispotent to suppress angiogenesis (Table 56) In A549 and PC3M resultsshowed also a significant reduction of bFGF. Protein amount of PIGF incell supernatants was only slightly but dose-dependently depressed inA549 and HTZ-19 cells. In PC3M cells basic endogenous PIGF level washigher than in all other tested cells and ASO effect was also stronger(Table 57). Finally, downregulation of Fit-1 protein in HT-29 cells(Table 58) and Tie-2 depression in HTZ-19 (ASO Seq. ID No. 218b 2.5 μM)and MCF-7 (mamma-carcinoma, 10 μM) could be detected (Table 59).

TABLE 54 mRNA expression of PAI-1 12 days after gymnotic transfer ofSeq. ID No. 218b in A549, HPAFII, HT-29, HTZ-19, TMK-1 and THP-1 cells.Regulation of PAI-1 gene expression is dose-dependently affected by ASOSeq. ID No. 218b in a manner for an improved disease prognosis. mRNAlevels were determined relative to housekeeping gene GNB2L1 usingquantitative real-time RT-PCR and then normalized to untreated control.Target PAI-1 mRNA levels 12 days after repeated gymnotic transfer (4 ×72 h) Cell line A549 HPAFII HT-29 HTZ-19 TMK-1 THP-1 n = 3 n = 1 n = 2 n= 2 n = 2 n = 2 A 1.00 ± 1.00 1.00 ± 1.00 ± 1.00 ± 1.00 ± 0.10 0.11 0.210.06 0.11 B 2.5 μM 1.28 ± 1.48 0.88 ± 0.99 ± 0.89 ± 1.14 ± 0.03 0.270.34 0.04 0.79 B 10 μM 1.03 ± 1.05 0.81 ± 1.30 ± 1.16 ± 1.21 ± 0.27 0.080.00 0.00 0.37 C 2.5 μM 0.91 ± 0.62 0.60 ± 1.13 ± 0.56 ± 0.83 ± 0.280.13 0.10 0.04 0.20 C 10 μM 0.56 ± 0.32 0.50 ± 0.77 ± 0.45 ± 0.09 ± 0.130.18 0.10 0.23 0.02 A = untreated control, B = Ref. 1, C = Seq. ID No.218b, ± = SEM, Statistics was calculated using theOrdinary-one-way-ANOVA followed by “Tukey's” multiple post hoccomparisons.

TABLE 55 VEGF protein levels in cell supernatant 12 days after gymnotictransfer of Seq. ID No. 218b in A549, HPAFII, HTZ-19, PC3M cells byMesoScale Discovery ® Assay (MSD Mesoscale Discovery, #K15198G). Proteinlevels were determined by measuring electro-chemiluminescence. TargetVEGF protein (pg/ml) 12 days after repeated gymnotic transfer (4 × 72 h)Cell line A549 HPAFII HTZ-19 PC3M n = 1 n = 2 n = 2 n = 2 A 8186 23266 ±876 4411 ± 66 2657 ± 103 B 2.5 8387  22278 ± 5711 3385 ± 57 1993 ± 5.4 μM B 10 8623 20776 ± 497 4044 ± 21  813 ± 0.8 μM C 2.5 8846 15479**++ ±512     3444 ± 197 1266*+ ± 20.5   μM C 10 6842 11214** ± 898  2882 ± 90442** ± 14.3  μM A = untreated control, B = Ref. 1, C = Seq. ID No.218b, ± = SEM, *p < 0.05 and **p < 0.01 in reference to A, +p < 0.05 and++p < 0.01 in reference to B 2.5 μM. Statistics were calculated usingthe Ordinary-one-way-ANOVA followed by “Tukey{acute over ( )}s” multiplepost hoc comparisons.

TABLE 56 bFGF protein levels in cell supernatant 12 days after gymnotictransfer of Seq. ID No. 218b in A549 and PC3M cells by MesoScaleDiscovery ® Assay (MSD Mesoscale Discovery, #K15198G). Protein levelswere determined by measuring electro-chemiluminescence. Target bFGFprotein (pg/ml) 12 days after repeated gymnotic transfer (4 × 72 h) Cellline A549 PC3M n = 2 n = 2 A 50.7 ± 2.9 21.2 ± 0.2 B 2.5 μM 54.4 ± 3.116.8 ± 0.1 B 10 μM 51.8 ± 2.7 14.7 ± 0.2 C 2.5 μM 26.7**++ ± 2.1  11.3**+ ± 0.0   C 10 μM 24.2 ± 3.4 7.6**++ ± 0.0   A = untreatedcontrol, B = Ref. 1, C = Seq. ID No. 218b, ± = SEM, *p < 0.05 and **p <0.01 in reference to A, +p < 0.05 and ++p < 0.01 in reference to B 2.5μM, #p < 0.05 and ##p < 0.01 in reference to B 10 μM. Statistics wascalculated using the Ordinary-one-way-ANOVA followed by “Tukey{acuteover ( )}s” multiple post hoc comparisons.

TABLE 57 PIGF protein levels in cell supernatant 12 days after gymnotictransfer of Seq. ID No. 218b in A549, HTZ-19 and PC3M cells by MesoScaleDiscovery ® Assay (MSD MesoScale Discovery ® , #K15198G). Protein levelswere determined by measuring electro-chemiluminescence. Target PIGFprotein (pg/ml) 12 days after repeated gymnotic transfer (4 × 72 h) Cellline A549 HTZ-19 PC3M n = 2 n = 1 n = 2 A 9.9 ± 0.4 11.6 61.7 ± 2.1 B2.5 μM 9.6 ± 0.2 8.1 54.1 ± 1.9 B 10 μM 8.6 ± 0.1 8.4 59.5 ± 3.2 C 2.5μM 8.2 ± 0.8 8.2 69.4 ± 2.4 C 10 μM 6.3** ± 0.9  6.5 45.0 ± 3.5 A =untreated control, B = Ref. 1, C = Seq. ID No. 218b, ± = SEM, **p < 0.01in reference to A, Statistics was calculated using theOrdinary-one-way-ANOVA followed by “Tukey{acute over ( )}s” multiplepost hoc comparisons

TABLE 58 Flt-1 protein levels in cell supernatant 12 days after gymnotictransfer of Seq. ID No. 218b in HTZ-19 cells by MesoScale Discovery ±assay (MSD Mesoscale Discovery, #K15198G). Protein levels weredetermined by measuring electro-chemiluminescence. Target Flt-1 protein(pg/ml) 12 days after repeated gymnotic transfer (4 × 72 h) Cell lineHT-29 n = 1 A 33.9 B 2.5 μM 27.7 B 10 μM 27.7 C 2.5 μM 18.2 C 10 μM 18.7A = untreated control, B = Ref. 1, C = Seq. ID No. 218b, ± = SEM, **p <0.01 in reference to A, Statistics was calculated using the Ordinaryone-way-ANOVA followed by “Tukey{acute over ( )}s” multiple post hoccomparisons.

TABLE 59 shows Tie-2 protein levels in cell supernatant 12 days aftergymnotic transfer of Seq. ID No. 218b in HTZ-19 and MCF-7 cells byMesoScale Discovery ± Assay (MSD Mesoscale Discovery, #K15198G). Proteinlevels were determined by measuring electro-chemiluminescence. TargetTie-2 protein (pg/ml) 12 days after repeated gymnotic transfer (4 × 72h) Cell line HTZ-19 MCF-7 n = 1 n = 1 A 13.5 98.1 B 2.5 μM 6.2 B 10 μM149.2 C 2.5 μM 3.2 C 10 μM 76.9 Protein levels were determined bymeasuring electro-chemiluminescence. A = untreated control, B = Ref. 1,C = Seq. ID No. 218b, ± = SEM, **p < 0.01 in reference to A, Statisticswas calculated using the Ordinary-one-way-ANOVA followed by “Tukey{acuteover ( )}s” multiple post hoc comparisons.

Conclusion

All analyzed pro-angiogenic factors (VEGF, bFGF, PIGF, Fit-1 and Tie-2)could be regulated by ASO Seq. ID No. 218b in a manner that would have afavorable impact on suppressing tumor progression and other pathologicalmechanisms dependent on enhanced angiogenesis. Furthermore, PAI-1 mRNAwas dose-dependently reduced by ASO Seq. ID No. 218b. This factor, aTGF-β target gene and e.g. an approved prognostic marker in breastcancer, was also dose-dependently downregulated.

Taken together, all tested inventive ASOs were efficient in reducingangiogenic processes that favors tumor progression, metastasis,inflammation, and thrombosis. Thus, the inventive ASOs directed againstTGF-R_(II) are potent therapeutic candidate in different types of cancerand thrombosis related diseases.

Example 20: Analysis of the Effect of Inventive ASOs Upon Fibrosis

TGF-β is involved in a lot of processes such as cell proliferation,migration, wound healing, angiogenesis and cell-cell interactions. It'sknown from several studies, that this factor is often elevated duringpathogenesis in several diseases including primary open angle glaucoma,Alzheimer disease, pulmonal fibrosis and diabetic nephropathy. Thesediseases are related to pathologic modifications in extracellular matrix(ECM) and the aktin-cytoskeleton. Often, these observed alterationscorrelate with severity disease progression and resistance to treatment(Epithelial Mesenchymal transition—EMT—in tumors). Connective tissuegrowth factor (CTGF) is a downstream-mediator of TGF-β and mediatesfibrotic effects of TGF-β. Thus, it is shown that CTGF mediatesdeposition of ECM and modulates reorganization of aktin-cytoskeleton. Toinvestigate whether the inventive ASOs contribute to a resolution offibrotic processes by inhibiting TGF-β signaling, CTGF levels wereevaluated in addition to fibronectin (FN) and Collagen IV (ColIV), whichrepresent two main components of ECM in several different cancer cells.Furthermore, effects of ASOs on CTGF, FN and on aktin-cytoskeleton wereexamined in neural precursor (ReNcell CX) and human lung cancer (A549)cells.

20.1 Fibrosis in Neurodegeneration

Description of Methods

Cells were cultured as described before in standard protocol. Fortreatment, cells were seeded in 24-well culture dishes (Sarstedt#83.1836.300) (50,000 cells/well), 6-well culture dishes (Sarstedt#83.3920.300) (80,000 cells/well) and 8-well cell culture slide dishes(Sarstedt #94.6140.802) (10,000 cells/well) and were incubated overnightat 37° C. and 5% CO₂. To investigate a response of ReNcell CX® cells toTGF-β1 cells were treated after refreshing of medium with TGF-β1 (2 and10 ng/ml, PromoCell #C₆₃₄₉₉) for 48 h, followed by mRNA analysis forCTGF. To figure out the ASO effect on CTGF and FN, ReNcell CX® cells,medium was removed and replaced by fresh full medium (1 ml for 6-welland 0.5 ml for 8-well). Ref. 1 (Scrambled control), ASO Seq. ID No. 218band Seq.ID No. 218b were then added in medium at concentrations of 2.5and 10 μM and respective analysis (real-time RT-PCR, Western Blotanalysis and Immunocytochemistry) was performed after 96 h. To examinethe ASO impact after investigation of pre-incubation with TGF-β1, mediumwas removed and replaced by fresh full medium (1 ml for 6-well dishesand 8-well cell culture slide dishes). Following exposition of TGF-β1(10 ng/ml, 48 h) medium was changed, TGF-β1 (10 ng/ml), Ref.1 (10 μM),ASO with Seq. ID No. 218b (10 μM) and ASO with Seq. ID No. 218c (10 μM)were added in combination and in single treatment to cells. ReNcell CX®cells were then harvested 96 h after gymnotic transfer. Therefore, cellswere washed twice with PBS and subsequently used for RNA (24-welldishes) and protein isolation (6-well dishes) or immunocytochemicalexamination of cells (in 8-well cell culture slide dishes). Protocols,antibodies, dilutions and primers were used as described before.

20.1.1 Results of TGF-β1 Effects on Neural Precursor Cells (ReNcell CX)

Nothing was known about reaction of ReNcell CX® to TGF-β1 exposure. ThusReNcell CX® cells were treated for 48 h with TGF-β1 in two differentconcentrations (Table 60). Evaluation of real-time RT-PCR revealed adose-dependent induction of CTGF- and TGF-β1 gene expression.

TABLE 60 CTGF and TGF-β1 mRNA expression 48 h after stimulation withTGF- β1. mRNA levels were determined relative to housekeeping geneGNB2L1 using quantitative real-time RT-PCR and then normalized tountreated control. Cell line ReNcell CX mRNA levels after 48 h TGF-β1treatment Target CTGF TGF-β1 Time point 48 h 48 h n = 3 n = 3 A 1.00 ±0.43 1.00 ± 0.10 E 2 ng/ml 1.73 ± 0.92 1.34 ± 0.45 E 10 ng/ml 2.15 ±1.14 1.85 ± 0.65 A = untreated control, E = TGF-β1. ± = SEM, Statisticswas calculated using the Ordinary-one-way-ANOVA followed by “Tukey{acuteover ( )}s” multiple post hoc comparison.

Conclusion

ReNcell CX® cells showed a response to TGF-β1 exposure presentingself-induction of TGF-β1 and elevation of TGF-β1 target gene CTGF. Takentogether, ReNcell CX® cells are ideal to examine questions addressingTGF-β effects.

20.1.2 Results for Seq. ID No. 218b

20.1.2.1 Effects of Gymnotic Transfer

Gymnotic transfer of ASO Seq. ID No. 218b results in a dose-dependentand significant reduction of CTGF and FN (Table 61). This impact of ASOSeq. ID No. 218b was verified for FN protein level. FN protein level wasdepressed by about 70% 96 h after gymnotic transfer of tested ASO,whereas TGF-β1 treatment of ReNcell CX® cells resulted in a 3.4-foldinduction of FN (Table 62).

TABLE 61 Dose-dependent and significant downregulation of CTGF mRNAafter gymnotic transfer with Seq. ID No. 218b in ReNcell CX ® cells.mRNA levels were determined relative to housekeeping gene GNB2L1 usingquantitative real-time RT-PCR and then normalized to untreated control.Cell line ReNcell CX mRNA levels after gymnotic transfer Target CTGF FNTime point 96 h, n = 3 96 h, n = 3 A 1.00 ± 0.04 1.00 ± 0.00 B 2.5 μM0.97 ± 0.06 0.81 ± 0.14 B 10 μM 0.86 ± 0.17 0.67 ± 0.07 C 2.5 μM 0.66**± 0.02  0.59 ± 0.02 C 10 μM 0.52** ± 0.02  0.39* ± 0.03  A = untreatedcontrol, B = Ref. 1, C = Seq. ID No. 218b. ± = SEM, *p < 0.05, **p <0.01 in reference to A. Statistics was calculated using theOrdinary-one-way-ANOVA followed by “Dunnett's” post hoc comparisons.

TABLE 62 Densitometric analysis after Western Blotting for Fibronectin.Downregulation of FN protein 96 h after gymnotic transfer of ASO Seq. IDNo. 218b in ReNcell OX ® cells could be recognized. Protein level wasdetermined relative to housekeeping gene alpha-Tubulin using ImageStudio ™ Lite Software and was then normalized to untreated control.Cell line ReNcell CX protein levels after gym notic transfer Target FNTime point 96 h, n = 1 A 1.00 B 2.5 μM 1.06 B 10 μM 0.60 C 2.5 μM 0.46 C10 μM 0.30 E 10 ng/ml 3.43 A = untreated control, B = Ref. 1, C = Seq.ID No. 218b.

Conclusion

ASO Seq. ID No. 218b was potent in downregulating mRNA levels of CTGFand FN in human neuronal precursor cells. ASO Seq. ID No. 218b treatmentreduced FN protein, 96 h after gymnotic transfer. Thus, TGF-R_(II)specific ASO mediates blocking of TGF-β induced fibrotic effects ReNcellCX® cells.

20.1.2.2 Effects of Gymnotic Transfer after TGF-β Pre-Incubation

To analyze whether ASO Seq. ID No. 218b is also potent in inhibitingfibrotic effects mediated by TGF-β under pathological conditions,ReNcell CX® cells were pre-incubated with TGF-β pre-incubation followedby gymnotic transfer for 96 h. Afterwards, determined mRNA levels ofCTGF and FN indicate a strong anti-fibrotic effect of ASO Seq. ID No.218b also after TGF-β induction of CTGF and FN gene expression (Table63). Immunocytochemical staining for CTGF (FIG. 25A) and FN (FIG. 25B)confirmed data from mRNA analysis. In addition, staining with phalloidinfor analysis of actin-cytoskeleton showed an induction of stress-fibersafter TGF-β treatment, whereas ASO Seq. ID No. 218b was efficient inblocking TGF-β-mediated stress fiber induction (FIG. 25C).

TABLE 63 Downregulation of CTGF and FN mRNA after TGF-β1-pre-incubationfollowed by gymnotic transfer with Seq. ID No. 218b in ReNcell CX ®cells (compared to scrambled control). mRNA levels were determinedrelative to housekeeping gene GNB2L1 using quantitative real-time RT-PCRand was then normalized to untreated control. Cell line ReNcell CX mRNAlevels after 48 h TGF-β1 -> 96 h TGF-β1 + ASOs/single treatment TargetCTGF FN Time point 96 h, n = 3 96 h, n = 3 A 1.00 ± 0.04 1.00 ± 0.10 B10 μM 0.85 ± 0.01 0.78 ± 0.20 C 10 μM 0.70* ± 0.25  0.44 ± 0.04 E 10ng/ml 1.60** ± 0.15  2.25 ± 0.31 E 10 ng/ml + B 10 μM 1.71** ± 0.03 4.08*++ ± 0.90   E 10 ng/ml + C 10 μM 1.19++ ± 0.04  1.74++ ± 0.61  A =untreated control, B = Ref. 1, C = Seq. ID No. 218b, E = TGF-β, ± = SEM,*p < 0.05, **p < 0.01 in reference to A. Statistics was calculated usingthe Ordinary-one-way-ANOVA followed by “Dunnett's” post hoc comparisons.

Conclusion

ASO Seq. ID No. 218b showed strong anti-fibrotic effects under simulatedpathological conditions (TGF-β1 pre-incubation). Aside fromdownregulation of FN as one main component of ECM, actin-cytoskeletonwas also affected by inventive ASO in a manner that may be beneficialfor a better outcome in fibrotic diseases.

20.1.3 Results for Seq. ID No. 218c

20.1.3.1 Effects of Gymnotic Transfer

Gymnotic transfer of ASO Seq. ID No. 218c results in a strong andsignificant reduction of CTGF mRNA after gymnotic transfer of 10 μM ASOSeq. ID No. 218c (Table 64).

TABLE 64 Downregulation of CTGF mRNA after gymnotic transfer of Seq. IDNo. 218c in ReNcell CX ± cells. mRNA levels were determined relative tohousekeeping gene GNB2L1 using quantitative real-time RT-PCR and thennormalized to untreated control. Cell line ReNcell CX mRNA levels aftergymnotic transfer Target CTGF Time point 96 h, n = 3 A 1.00 ± 0.10 B 2.5μM 0.88 ± 0.08 B 10 μM 0.89 ± 0.07 D 2.5 μM 0.48 ± 0.08 D 10 μM 0.17* ±0.02  A = untreated control, B = Ref. 1, D = Seq. ID No. 218c. ± = SEM,*p < 0.05 in reference to A. Statistics was calculated using theOrdinary-one-way-ANOVA followed by “Dunnett's” post hoc comparisons.

Conclusion

ASO Seq. ID No. 218c was efficient in dose-dependent reduction of CTGFmRNA.

20.1.3.2 Effects of Gymnotic Transfer after TGF-R Pre-Incubation

Results for gymnotic transfer for ASO Seq. ID 218c followed by TGF-β1pre-incubation verified an effective blockage of TGF-β1 induced effectson CTGF mRNA levels (Table 65). ASO was such potent in blocking TGF-β1effect on CTGF that combination treatment is comparable to ASO Seq. IDNo. 218c single treatment.

TABLE 65 CTGF mRNA level after TGF-β1 pre-incubation following gymnotictransfer of Seq. ID No. 218c and parallel TGF-β1 treatment in ReNcellCX ® cells. Data confirmed an effective blocking of TGF-β1 inducedeffects on CTGF mRNA level by ASO Seq. ID No. 218c in comparison tocombination treatments. mRNA levels were determined relative tohousekeeping gene GNB2L1 using quantitative real-time RT-PCR and thennormalized to untreated control. Target ReNcell CX Time point mRNAlevels 48 h TGF-β1 −> 96 h TGF-β1 + ASOs/single treatment Cell line CTGFn = 3 A 1.00 ± 0.03 B 10 μM 0.85 ± 0.01 D 10 μM 0.17* ± 0.02  E 10 ng/ml1.39 ± 0.08 E 10 ng/ml + B 10 μM 1.25 ± 0.44 E 10 ng/ml + D 10 μM 0.23*± 0.02  A = untreated control, B = Ref. 1, D = Seq. ID No. 218c, E =TGF-β1. ± = SEM, *p < 0.05 in reference to A. Statistics was calculatedusing the Ordinary-one-way-ANOVA followed by “Dunnett's” post hoccomparisons.

Conclusion

ASO Seq. ID No. 218c showed a strong downregulation of CTGF mRNA andprotein even under artificial pathological conditions (TGF-β1pre-incubation).

Taken together, aside from strong anti-fibrotic effects, TGF-R_(II)specific ASOs showed a modulation of actin-cytoskeleton. Induction ofstress fibers may cause an elevation of cell rigidity and stiffness thatmay play a role e.g. in Alzheimer disease and other NeurodegenerativeDisorders. ECM deposition may also mediate fast pathogenic modificationse.g. in primary open angle glaucoma. Thus, reduction of ECM depositionand suppression of stress fiber formation may be profitable for a betterprognosis in fibrotic related neurological disorders. Thereby,TGF-R_(II) specific ASOs are potent therapeutic agents for the treatmente.g. Alzheimer disease and primary open angle glaucoma.

20.2. Pulmonary Fibrosis

Description of Methods

For investigation of ASO effects to ECM and actin-cytoskeleton in lung,human lung cancer (A549) cells were examined and cultured as describedbefore. For treatment, cells were seeded in 24-well culture dishes(Sarstedt #83.1836.300) (50,000 cells/well), 6-well culture dishes(Sarstedt #83.3920.300) (80,000 cells/well) and 8-well cell cultureslide dishes (Sarstedt #94.6140.802) (10,000 cells/well) and wereincubated overnight at 37° C. and 5% CO₂. To investigate a response ofA549 cells to TGF-β1 cells were treated after refreshing of medium withTGF-β1 (2 and 10 ng/ml, PromoCell #C₆₃₄₉₉) for 48 h following mRNAanalysis for CTGF. To investigate the ASO effect on CTGF and FN A549cells, medium was removed and replaced by fresh full medium (1 ml for6-well and 0.5 ml for 8-x-well). Ref. 1 (scrambled control), ASO Seq. IDNo. 218b and Seq.ID No. 218b were then added in medium at concentrationsof 2.5 and 10 μM and respective analysis (real-time RT-PCR, Western Blotanalysis and Immunocytochemistry) was performed after 72 h in ReNcellCX® cells. To show possible ASO impact after pre-incubation with TGF-β1,medium was removed and replaced by fresh full medium (1 ml for 6-welldishes and 8-well cell culture slide dishes). Following exposition ofTGF-β1 (10 ng/ml, 48 h) medium was changed, TGF-β1 (10 ng/ml), Ref.1 (10μM), ASO with Seq. ID No. 218b (10 μM) and ASO with Seq. ID No. 218c (10μM) was added in combination and in single treatment to cells. A549cells were then harvested 72 h after gymnotic transfer. Therefore, cellswere washed twice with PBS and subsequently used for RNA (24-welldishes) and protein isolation (6-well dishes) or immunocytochemicalexamination of cells (in 8-well cell culture slide dishes). Protocols,used antibodies, dilutions and primers were as described before.

20.2.1 Results of TGF-β1 Effects on Lung Cancer Cells (A549)

To investigate the ability of A549 cells to react to TGF-β1 exposure,cells were treated for 48 h with TGF-β1 in two different concentrations(Table 66). Evaluation of real-time RT-PCR revealed for CTGF and TGF-β1itself a dose-dependent induction of gene expression.

TABLE 66 Induced CTGF and TGF-β1 mRNA expression 48 h after stimulationwith TGF-β1 in A549 cells. mRNA expression levels were determinedrelative to housekeeping gene GNB2L1 using quantitative real- timeRT-PCR and then normalized to untreated control. Cell line A549 mRNAlevels after 48 h TGF-β1 treatment Target CTGF TGF-β1 Time point 48 h, n= 3 48 h, n = 3 A     1.00 ± 0.23 1.00 ± 0.31 E 2 ng/ml    2.44* ± 0.181.60 ± 0.34 E 10 ng/ml 11.35**++ ± 0.52 2.37 ± 0.36 A = untreatedcontrol, E = TGF-β1. ± = SEM, *p < 0.05 and **p < 0.01 in reference toA, ++p < 0.05 in reference to E 2 ng/ml. Statistics was calculated usingthe Ordinary-one-way-ANOVA followed by “Tukey's” multiple post hoccomparison.

Conclusion

A549 cells showed a dose-dependent and significant mRNA upregulation ofCTGF upon TGF-β1 exposure. In addition, self-induction of TGF-β1 wasobserved. Taken together, A549 cells are a good model to examinequestions addressing TGF-β effects in lung and lung cancer.

20.2.2 Results for Seq. ID No. 218b

20.2.2.1 Results for Effects of Gymnotic Transfer

Gymnotic transfer of ASO Seq. ID No. 218b causes a dose-dependent andhighly significant reduction of CTGF gene expression (Table 67). FN mRNAlevel was also affected by tested ASO but not dose-dependently. Incontrast, staining against FN revealed a dose-dependent reduction of FNin comparison to scrambled control (FIG. 260A). Furthermore, ASO andTGF-β impact on actin-cytoskeleton was examined. FIG. 26B showed aninduction of actin-fibers including stress-fiber formation after TGF-β1treatment in A549 cells in doss-dependent manner, whereas signal aftergymnotic transfer of ASO Seq. ID No. 218b in A549 cells wassignificantly downregulated parallel to recognized reversion of TGF-β1-mediated effects. For protein analysis a proper downregulation of CTGFparallel to an inhibition of pErk1/2 by which CTGF mediates its fibroticeffects could have been shown (Table 68). Furthermore, 72 h aftergymnotic transfer of ASO Seq. ID No. 218b a decrease of both ECM maincomponents FN and ColIV was remarkable (Table 68).

TABLE 67 Dose-dependent and significant downregulation of CTGF mRNAafter gymnotic transfer with Seq. ID No. 218b in A549 cells. mRNA levelswere determined relative to housekeeping gene GNB2L1 using quantitativereal-time RT-PCR and then normalized to untreated control. Cell lineA549 mRNA levels after gymnotic transfer Target CTGF FN Time point 72 h,n = 3 72 h, n = 3 A 1.00 ± 0.08 1.00 ± 0.07 B 2.5 μM 0.87 ± 0.06 1.08 ±0.02 B 10 μM 0.80 ± 0.03 0.87 ± 0.08 C 2.5 μM 0.60** ± 0.04  0.77 ± 0.17C 10 μM 0.39** ± 0.03  0.74 ± 0.16 A = untreated control, B = Ref. 1, C= Seq. ID No. 218b. ± = SEM, **p < 0.01 in reference to A. Statisticswas calculated using the Ordinary-one-way-ANOVA followed by “Dunnett's”post hoc comparisons.

TABLE 68 Densitometric analysis after CTGF, FN, ColIV and pErk11/2Western Blot: 72 h after gymnotic transfer with ASO Seq. ID No 218b inA549. Protein level was determined relative to housekeeping genealpha-Tubulin using Image Studio ™ Lite Software and was then normalizedto untreated control. Cell line A549 protein levels after gymnotictransfer Target CTGF FN ColIV pErk1/2 Time point 72 h 72 h 72 h 72 h n =1 n = 1 n = 1 n = 2 A 1.00 1.00 1.00 1.00 ± 0.00 B 2.5 μM 0.91 0.89 1.191.00 ± 0.14 B 10 μM 1.31 0.76 0.87 0.98 ± 0.02 C 2.5 μM 0.05 0.81 1.160.67 ± 0.26 C 10 μM 0.09 0.46 0.65 0.61 ± 0.13 A = untreated control, B= Ref. 1, C = Seq. ID No. 218b.

Conclusion

Gymnotic transfer of Seq. ID No. 218b was efficient in modulatingfactors which are involved in ECM deposition and actin-cytoskeletonreorganization in human lung cells.

20.2.2.2 Results for Effects of Gymnotic Transfer after TGF-β1Pre-Incubation

Results for gymnotic transfer of ASO Seq. ID 218b following TGF-β1pre-incubation verified an effective blockage of strong TGF-β1 inducedeffects on CTGF and FN mRNA levels (Table 69). Immunocytochemicalstaining against CTGF (FIG. 27A) and FN (FIG. 27B) confirmed mRNAdetection on protein level.

TABLE 69 CTGF and FN mRNA level after TGF-β1-pre-incubation followinggymnotic transfer of Seq. ID No. 218b and parallel TGF-β1 treatment inA549 cells. Data confirmed an effective blocking of TGF-β1 inducedeffects on CTGF and FN mRNA levels by ASO Seq. ID No. 218b in comparisonto combination treatments. mRNA levels were determined relative tohousekeeping gene GNB2L1 using quantitative real-time RT-PCR and thennormalized to untreated control. Target A549 Time point mRNA levels 48 hTGF-β1 −> 72 h TGF-β1 + ASOs/single treatment Cell line CTGF FN n = 5 n= 3 A 1.00 ± 0.22 1.00 ± 0.45 B 10 μM 0.89 ± 0.19 1.02 ± 0.37 C 10 μM0.52 ± 0.05 0.35 ± 0.06 E 10 ng/ml 6.92* ± 2.32  2.92 ± 1.02 E 10ng/ml + B 10 μM 8.79** ± 2.72  2.90 ± 0.56 E 10 ng/ml + C 10 μM 2.53 ±0.59 1.18 ± 0.28 A = untreated control, B = Ref. 1, C = Seq. ID No.218b, E = TGF-β1. ± = SEM, *p < 0.05, **p < 0.01 in reference to A.Statistics was calculated using the Ordinary-one-way-ANOVA followed by“Dunnett's” post hoc comparisons.

Conclusion

ASO Seq. ID No. 218b was efficient in mediating anti-fibrotic effects inA549 cells under artificial pathological conditions mimicked excessiveconcentrations of TGF-β1.

20.2.3 Results for Seq. ID No. 218c

20.2.3.1 Results for Effects of Gymnotic Transfer

Gymnotic transfer of ASO Seq. ID No. 218c mediates a strongdose-dependent and significant reduction of CTGF mRNA 72 h aftergymnotic transfer in A549 cells (Table 70).

TABLE 70 Downregulation of CTGF mRNA 72 h after gymnotic transfer ofSeq. ID No. 218c in A549 cells. mRNA levels were determined relative tohousekeeping gene GNB2L1 using quantitative real- time RT-PCR and thennormalized to untreated control. Cell line A549 mRNA level aftergymnotic transfer Target CTGF Time point 72 h n = 4 A 1.00 ± 0.08 B 2.5μM 0.97 ± 0.07 B 10 μM 0.85 ± 0.06 D 2.5 μM 0.49** ± 0.05  D 10 μM0.31** ± 0.03  A = untreated control, B = Ref. 1, D = Seq. ID No. 218c.± = SEM, **p < 0.01 in reference to A. Statistics were calculated usingthe Ordinary-one-way-ANOVA followed by “Dunnett's” post hoc comparisons.

Conclusion

Gymnotic transfer of ASO Seq. ID No. 218c was efficient in reducing mRNAof TGF-β downstream-mediator CTGF.

20.2.2.2 Results for Effects of Gymnotic Transfer after TGF-RPre-Incubation

Results for gymnotic transfer for ASO Seq. ID No. 218c following TGF-β1pre-incubation verified an effective blockage of strong TGF-β1 inducedeffects on CTGF mRNA levels (Table 71). Immunocytochemical stainingagainst CTGF confirmed these findings on protein level (FIG. 28).

TABLE 71 CTGF mRNA levels after TGF-β1 pre-incubation followed bygymnotic transfer of Seq. ID No. 218c and parallel TGF-β1 treatment inA549. Data verified an effective blockage of TGF- β1 induced effects onCTGF mRNA levels by ASO Seq. ID No. 218c in comparison to combinationtreatments. mRNA levels were determined relative to housekeeping geneGNB2L1 using quantitative real-time RT-PCR and then normalized tountreated control. Target A549 Time point 48 h TGF-β1 −> 72 h TGF-β1 +ASOs/single treatment Cell line CTGF n = 3 A 1.00 ± 0.05 B 10 μM 0.86 ±0.11 D 10 μM 0.53 ± 0.10 E 10 ng/ml 4.71 ± 1.76 E 10 ng/ml + B 10 μM5.89* ± 2.16  E 10 ng/ml + D 10 μM 0.86++ ± 0.06   A = untreatedcontrol, B = Ref. 1, D = Seq. ID No. 218c, E = TGF-β1. ± = SEM, **p <0.01 in reference to A, ++p < 0.01 in reference to E + B. Statistics wascalculated using the Ordinary-one-way-ANOVA followed by “Tukey's” posthoc comparisons.

Conclusion

ASO Seq. ID 218c was potent in mediating anti-fibrotic effects in A549cells under artificial pathological conditions mimicked by excessiveTGF-β1 concentrations. Taken together, ASO Seq. ID 218c is an effectivetherapeutic agent, because pathology of lung fibrosis could be sloweddown by reducing CTGF, FN and ColIV. In addition, stress fiber formationcan be reduced effectively by TGF-R_(II) specific ASO, making inventiveASOs ideal therapeutic agents.

20.3 Effects on Several Cancer Cells

Description of Methods

For investigation of ASO effects addressing ECM (CTGF, FN, ColIV) cellswere used and cultured as described before in standard protocol (Table10). For treatment, cells were seeded in 24-well culture dishes(Sarstedt #83.1836.300) (30,000 cells/well), 6-well culture dishes(Sarstedt #83.3920.300) (50,000 cells/well) and were incubated overnightat 37° C. and 5% CO₂. To analyze mRNA expression and influence on CTGF,FN and ColIV mRNA and protein levels cells were treated with Ref.1(Scrambled control) or ASO Seq. ID No. 218b at concentrations of 2.5 and10 μM and were incubated for 72 h at 37° C. and 5% CO₂. Treatmentincluding medium replacement was repeated 3 times every 72 h (12 days intotal). For harvesting, cells were washed twice with PBS andsubsequently used for RNA isolation (24-well dishes) or proteinisolation (6-well dishes). Protocols for RNA and protein isolation aswell as used antibodies and dilutions were performed as described above.

20.3.1 Results for Seq. ID No. 218b

Anti-fibrotic effects were detected by analysis of CTGF, FN, ColIV mRNAand protein levels. CTGF mRNA (Table 72) was dose-dependently reduced bySeq. ID No. 218b in HT-29, HTZ-19, MCF-7 and THP-1 cells. For KG-1 cellsdownregulation of TGF-β downstream-mediator was recognized for 2.5 μMASO Seq. ID No. 218b. For A549, Panc-1 and CaCo2 cells a decrease of FNwas demonstrated (Table 73) in accordance to a dose-dependently declineof ColIV mRNA (Table 74) in THP-1, HTZ-19 and L3.6pl cells (Table 65).Western Blot analysis revealed a strong reduction of CTGF protein inHT-29, MCF-7, TMK-1 and L3.6pl cells. Result for MCF-7 was significant(Table 75). In addition, phosphorylation of Erk1/2 in A549 and TMK-1cells was inhibited by ASO Seq. ID No. 218b. pErk1/2 is normallyactivated by CTGF to induce TGF-β mediated fibrotic effects (Table 76).For FN (A549, MCF-7, HT-29, HTZ-19, HPAFII) and Col IV (A549, HTZ-19,HPAFII, PC3M) (Table 77 and 78), the two main components of ECM, proteinlevels were minimized by about 50%.

TABLE 72 mRNA expression of CTGF 12 days after gymnotic transfer of Seq.ID No. 218b in HT-29, HTZ-19, KG1, MCF-7 and THP-1 cells. CTGF mRNA wasdecreased after gymnotic transfer of Seq. ID No. 218b for all testedcell lines. mRNA levels were determined relative to housekeeping geneGNB2L1 using quantitative real-time RT- PCR and then normalized tountreated control. Target CTGF mRNA levels 12 days after repeatedgymnotic transfer (4 × 72 h) Cell line HT-29 HTZ-19 KG-1 MCF-7 THP-1 n =2 n = 1 n = 1 n = 1 n = 2 A 1.00 ± 0.28 1.00 1.00 1.00 1.00 ± 0.28 B 2.5μM 0.68 ± 0.11 1.30 0.93 0.99 ± 0.68 B 10 μM 0.65 ± 0.03 1.20 0.88 0.911.15 ± 0.34 C 2.5 μM 0.40 ± 0.20 0.64 0.24 0.98 ± 0.11 C 10 μM 0.33 ±0.19 0.55 0.26 0.22 0.09 ± 0.03 A = untreated control, B = Ref. 1, C =Seq. ID No. 218b, ± = SEM, Statistics was calculated using theOrdinary-one-way-ANOVA followed by “Tukey's” multiple post hoccomparisons.

TABLE 73 mRNA expression of FN 12 days after gymnotic transfer of Seq.ID No. 218b in A549, Panc-1 and CaCo2 cells. FN mRNA was decreased aftergymnotic transfer of Seq. ID No. 218b for all tested cell lines. mRNAlevels were determined relative to housekeeping gene GNB2L1 usingquantitative real-time RT-PCR and then normalized to untreated control.Target FN mRNA levels 12 days after repeated gymnotic transfer (4 × 72h) Cell line A549 Panc-1 CaCo2 n = 2 n = 1 n = 2 A 1.00 ± 0.39 1.00 1.00± 0.30 B 2.5 μM 0.83 ± 0.08 1.29 0.55 ± 0.13 B 10 μM 1.00 ± 0.76 C 2.5μM 0.35 ± 0.20 0.15 0.73 ± 0.54 C 10 μM 0.18 ± 0.17 A = untreatedcontrol, B = Ref. 1, C = Seq. ID No. 218b, ± = SEM, **p < 0.01 inreference to A. Statistics was calculated using theOrdinary-one-way-ANOVA followed by “Tukey's” multiple post hoccomparisons.

TABLE 74 mRNA expression of ColIV 12 days after gymnotic transfer ofSeq. ID No. 218b in A549, HTZ-19, THP-1, L3.6pl, Panc-1 and CaCo2 cells.ColIV mRNA was decreased after gymnotic transfer of Seq. ID No. 218b forall tested cell lines. mRNA levels were determined relative tohousekeeping gene GNB2L1 using quantitative real- time RT-PCR and thennormalized to untreated control. Target Col IV mRNA levels 12 days afterrepeated gymnotic transfer (4 × 72 h) Cell line A549 THP-1 HTZ-19 L3.6plPanc-1 CaCo2 n = 2 n = 2 n = 1 n = 2 n = 1 n = 2 A 1.00 ± 1.00 ± 1.001.00 ± 1.00 1.00 ± 0.00 0.22 0.20 0.71 B 2.5 μM 1.18 ± 0.71 ± 0.94 0.83± 0.98 1.37 ± 0.31 0.25 0.09 0.19 B 10 μM 1.11 ± 0.61 ± 0.91 ± 0.57 2.61± 0.60 0.03 0.29 0.01 C 2.5 μM 0.84 ± 0.65 ± 0.51 1.14 ± 0.59 1.30 ±0.02 0.19 0.13 0.03 C 10 μM 0.75 ± 0.30 ± 0.69 ± 0.30 0.57 ± 0.02 0.130.05 0.14 A = untreated control, B = Ref. 1, C = Seq. ID No. 218b, ± =SEM, Statistics was calculated using the Ordinary-one-way-ANOVA followedby “Tukey's” multiple post hoc comparisons.

TABLE 75 Densitometric analysis after Western Blotting in HT-29, MCF-7,L3.6pl and TMK-1 cells 12 days after gymnotic transfer of Seq. ID No.218b. Downregulation of CTGF protein by ASO Seq. ID No. 218b could berecognized. Protein levels were determined relative to housekeeping genealpha-Tubulin using Image Studio ™ Lite Software and was then normalizedto untreated control. Target CTGF protein levels 12 days after repeatedgymnotic transfer (4 × 72 h) Cell line HT-29 MCF-7 TMK-1 L3.6pl n = 1 n= 2 n =1 n = 1 A 1.00    1.00 ± 0.0 1.00 1.00 B 10 μM 1.19    1.12 ±0.11 0.85 0.93 C 10 μM 0.50 0.22**++ ± 0.03 0.38 0.22 A = untreatedcontrol, B = Ref. 1, C = Seq. ID No. 218b. Statistics was calculatedusing the Ordinary-one-way-ANOVA followed by “Tukey's” multiple post hoccomparisons.

TABLE 76 Densitometric analysis after Western Blotting in A549 and TMK-1cells 12 days after gymnotic transfer of Seq. ID No. 218b.Downregulation of pErk1/2 protein by ASO Seq. ID No. 218b wasdetermined. Quantification of protein level was done relative tohousekeeping gene alpha-Tubulin using Image Studio ™ Lite Software andwas then normalized to untreated control. Target pErk1/2 protein levels12 days after repeated gymnotic transfer (4 × 72 h) Cell line A549 TMK-1n = 1 n =1 A 1.00 1.00 B 10 μM 1.21 1.14 C 10 μM 0.58 0.76 A = untreatedcontrol, B = Ref. 1, C = Seq. ID No. 218b. Statistics was calculatedusing the Ordinary-one-way-ANOVA followed by “Tukey's” multiple post hoccomparisons.

TABLE 77 Densitometric analysis after Western Blotting in A549, MCF-7,HT- 29, HTZ-19 and HPAFII cells 12 days after gymnotic transfer of Seq.ID No. 218b. Downregulation of FN protein by ASO Seq. ID No. 218b wasdetermined. Quantification of protein level was done relative tohousekeeping gene alpha-Tubulin using Image Studio ™ Lite Software andwas then normalized to untreated control. Target FN protein levels 12days after repeated gymnotic transfer (4 × 72 h) Cell line A549 MCF-7HT-29 HTZ-19 HPAFII n = 1 n = 2 n = 1 n = 1 n = 1 A 1.00 1.00 ± 0.221.00 1.00 1.00 B 10 μM 1.10 1.08 ± 0.25 0.81 1.20 1.12 C 10 μM 0.56 0.69± 0.18 0.40 0.83 0.56 A = untreated control, B = Ref. 1, C = Seq. ID No.218b. Statistics was calculated using the Ordinary-one-way-ANOVAfollowed by “Tukey's” multiple post hoc comparisons.

TABLE 78 Densitometric analysis after Western Blotting in A549, MCF-7,HT-29, HTZ-19 and HPAFII cells 12 days after gymnotic transfer of Seq.ID No. 218b. Downregulation of FN protein by ASO Seq. ID No. 218b wasdetermined. Protein levels were analyzed relative to housekeeping genealpha-Tubulin using Image Studio ™ Lite Software and was then normalizedto untreated control. Target Col IV protein levels 12 days afterrepeated gymnotic transfer (4 × 72 h) Cell line A549 HTZ-19 HPAFII PC3Mn = 1 n = 1 n = 1 n = 1 A 1.00 1.00 1.00 1.00 B 10 μM 1.31 1.01 1.051.07 C 10 μM 0.61 0.36 0.76 0.43 A = untreated control, B = Ref. 1, C =Seq. ID No. 218b. Statistics was calculated using theOrdinary-one-way-ANOVA followed by “Tukey's” multiple post hoccomparisons.

Conclusion

Increased deposition of ECM mediated by TGF-β1, through itsdownstream-mediator CTGF, could be efficiently reversed by TGF-R_(II)specific inventive ASOs in different tumor cell lines. A reduced levelof ECM components could contribute to a less aggressive in tumorprogression. Taken together, tested ASOs may demonstrate a newtherapeutic strategy in different fibrosis-associated diseases.

Example 21: Threshold for Toxicity of Inventive ASOs by ChronicIntracerebroventricular Administration Using a Dose-Escalation Paradigmin Cynomolgus

To evaluate the ideal dose range for the GLP-toxicity study, apre-experiment using chronic intracerebroventricular (icv)antisense-oligonucleotide (ASO) administration with escalating doses wasperformed in Cynomolgus monkeys. During the administration paradigmanimals were monitored for immunological, hematological andphysiological alterations.

Description of Method:

For chronic central ASO infusion in male and female Cynomolgus monkeys,a gas-pressure pump (0.25 ml/24 h, Tricumed-IP 2000V®) connected to asilicone catheter, targeting the right lateral ventricle was implantedsubcutaneously under ketamine/xylacin anesthesia and semi-sterileconditions. A single male and a single female monkey were used for eachtreatment condition (Seq. ID No. 218b, Seq. ID No. 218c, concentrationsgiven in Table 79). Each pump was implanted subcutaneously in theabdominal region via a 10 cm long skin incision at the neck of themonkey and was connected with the icy cannula by a silicone catheter.Animals were placed into a stereotaxic frame, and the icy cannula waslowered into the right lateral ventricle. The cannula was fixed with twostainless steel screws using dental cement (Kallocryl, Speiko®-Dr.Speier GmbH, Münster, Germany). The skin of the neck was closed withsutures. During surgery, the body temperature was maintained by aheating pad. To avoid post-surgical infections, monkeys were locallytreated with Betaisodona® (Mundipharma GmbH, Limburg, Germany) andreceived 1 ml antibiotics (sc, Baytril® 2.5% Bayer Vital GmbH,Leverkusen, Germany). The tubing and the resp. pump was filled with therespective treatment solution. ASO infusion periods (1 week for eachdose) were interrupted by a one-week wash out period with 0.9% NaClbeing administered exclusively. During the entire administrationparadigm body weight development and food consumption were monitored.Further, blood and CSF samples were taken once a week to determinehematological as well as immunological alterations but also systemic ASOconcentrations. On the last day, animals were sacrificed, and organs(liver, kidneys, brain) were removed, and analyzed for proliferation,apoptosis, mRNA knock down, and tumor formation.

TABLE 79 Experimental design and the doses of ASOs given during the7-week administration paradigm. Treatment condition Week 1 Week 2 Week 3Week 4 Week 5 Week 6 Week 7 Seq. ID No. 218b 0.048 mM 0.9% NaCl 0.24 mM0.9% NaCl 1.2 mM 0.9% NaCl 6 mM Seq. ID No 218c 0.048 mM 0.9% NaCl 0.24mM 0.9% NaCl 1.2 mM 0.9% NaCl 6 mM

Conclusion:

All tested, inventive ASOs were at least non-toxic in weeks 1-6 and weretherefore used for further research and toxicological examination.

Example 22: Determination of Behavioral and Physiological AbnormalitiesFollowing Central Antisense-Oligonucleotide Administration

The goal of this study was to investigate the effects of a singleintracerebroventricular (icv) antisense-oligonucleotide administrationon neurological and resulting behavioral parameters in rats.

Description of Method:

Stereotaxic procedures were performed under ketamine/xylacin anesthesiaand semi-sterile conditions. Following surgery, rats had two days forrecovery.

Implantation of Icy Guide Cannula

Animals were placed into a stereotaxic frame, and the guide cannula (12mm) was implanted 2 mm above the left lateral ventricle (coordinatesrelative to bregma: 1.0 mm posterior, -1.6 mm lateral to midline, 1.8 mmbeneath the surface of the skull. The guide cannula was anchored to twostainless steel screws using dental acrylic cement (Kallocryl,Speiko®-Dr. Speier GmbH, Monster, Germany) and was closed with a dummycannula. During surgery, the body temperature was maintained by aheating pad. To avoid post-surgical infections, mice were locallytreated with Betaisodona® (Mundipharma GmbH, Limburg, Germany) andreceived 0.1 ml antibiotics (sc, Baytril® 2.5% Bayer Vital GmbH,Leverkusen, Germany).

ICV Infusion

Slightly restrained rats received an icy infusion of either ASO (2 μM/5μl, 10 μM/5 μl, 50 μM/5 μl, 250 μM/5 μl) or vehicle (5 μl, 0.9% NaCl, pH7.4, Braun) using a 27-gauge cannula, which extended 2 mm beyond theguide cannula and remained in place for 30 s to allow diffusion. Ratswere monitored 15, 30, 60 and 120 minutes following icy administrationfor behavioral reactions, motor activity, CNS excitation, posture, motorcoordination, muscle tone, reflexes, and body temperature.

Verification of Cannula and Microdialysis Probe Placement

After scarification, brains were removed, snap frozen and stored at −80°C. until analyzation. Histological verification of the icy implantationsites was performed at 40-pm coronal, cresyl violet-stained brainsections.

The present results demonstrate a single ASO (for both sequences Seq. IDNo. 218b, Seq. ID No. 218c) icy administration, for different doses, tobe a safe and secure technique in rats due to no effects on neurologicalparameters.

Example 23: Determination of the Ideal Dose Range for the CynomolgusGLP-Toxicity Study (Pre-Toxicity Experiment in Rats)

To investigate any general toxicological effects of a daily intravenous(iv) antisense-oligonucleotide (ASO) administration, and to localize theperfect dose-range for the GLP-pre-toxicity study in rats, apre-toxicity experiment in rats was performed.

Description of Method:

For repeated intravenous ASO injection 20 male and 20 female rats weredivided into four treatment groups, a vehicle group, an ASO_(low), anASO_(intermediate), and an ASO_(high) group. This paradigm was performedfor Seq. ID No. 218b and Seq. ID No. 218c. Rats received a daily ivbolus ASO injection for 15 consecutive days. Rats were monitored formortality (twice daily), clinical symptoms (once daily, bod weightdevelopment (weekly), food consumption (weekly). On day 15 of theexperimental paradigm, animals were sacrificed, organs (liver, kidney,brain) were removed and trunk blood was collected. Afterwards tissuesand blood was analyzed for immunological and hematological alterations.

The results of the present study demonstrate the two ASOs Seq. ID No.218b and Seq. ID No. 218c to be a safe medication for a variety ofdifferent disorders with no toxic effects when administered at low andintermediate doses.

Example 24: Determination of any General Toxicological Effects byRepeated Intravenous Antisense-Oligonucleotide Injection

The goal of this study was to investigate at which dose a dailyintravenous (iv) antisense-oligonucleotide (ASO) administration exertsany general toxicological effects in rats.

Description of Method:

For repeated intravenous ASO injection 80 male and 80 female rats weredivided into four treatment groups, a vehicle group, an ASO_(low), anASO_(intermediate), and an ASO_(high) group. Rats received a daily ivbolus ASO injection for 29 consecutive days. Rats were monitored formortality (twice daily), clinical symptoms (once daily, bod weightdevelopment (weekly), food consumption (weekly). On day 29 of theexperimental paradigm, animals were sacrificed, organs (liver, kidney,brain) were removed and trunk blood was collected. In addition, bonemarrow smears were collected. Afterwards tissues and blood was analyzedfor immunological and hematological, and histopathological alterations.

The results of the present study demonstrate the two ASOs Seq. ID No.218b and Seq. ID No. 218c to be a safe medication for a variety ofdifferent disorders with no toxic effects when administered at low andintermediate doses.

Example 25: Determination of the Toxicological Properties of a ChronicCentral Antisense-Oligonucleotide Administration in Cynomolgus Monkeys

To determine the effective, and to identify the toxic dose, male andfemale Cynomolgus monkeys received different doses of an inventiveantisense-oligonucleotide (ASO) by chronic intracerebroventricularadministration. During the administration paradigm, animals weremonitored for immunological, hematological and physiologicalalterations.

Description of Method:

For chronic central ASO infusion in male and female Cynomolgus monkeys,a gas-pressure pump (0.25 ml/24 h, Tricumed IP-2000V®) connected to asilicone catheter, targeting the right lateral ventricle, was implantedsubcutaneously under ketamine/xylacin anesthesia and semi-sterileconditions. Three male and three female monkeys were used for eachtreatment condition (vehicle, ASO_(low), ASO_(high), concentrationsgiven in Table 79). Further, for investigating the timeframe forrecovery, two male and two female monkeys (vehicle, and ASO_(high)) weresacrificed four weeks after ASO administration was terminated. Each pumpwas implanted subcutaneously in the abdominal region via a 10 cm longskin incision at the neck of the monkey and connected with the icycannula by a silicone catheter. Animals were placed into a stereotaxicframe, and the icy cannula was lowered into the right lateral ventricle.The cannula was fixed with two stainless steel screws using dentalcement (Kallocryl, Speiko®-Dr. Speier GmbH, Münster, Germany). The skinof the neck was closed with sutures. During surgery, the bodytemperature was maintained by a heating pad. To avoid post-surgicalinfections, monkeys were locally treated with Betaisodona® (MundipharmaGmbH, Limburg, Germany) and received 1 ml antibiotics (sc, Baytril® 2.5%Bayer Vital GmbH, Leverkusen, Germany). The tubing was filled with therespective treatment solution. During the entire administration paradigmbody weight development and food consumption was monitored. Further,blood and aCSF samples were taken once a week to determine hematologicalas well as immunological alterations but also systemic ASOconcentrations. On the last day, animals of the main study weresacrificed, and organs (liver, kidneys, brain) were removed, andanalyzed for proliferation, apoptosis, mRNA knock down, and tumorformation. After week 57, the additional animals used for investigatingrecovery periods were also sacrificed and the same read out parameterswere determined.

TABLE 80 Treatment conditions and the animals per group for the 4-weekGLP- toxicity study and for the additional 4-week recovery period.Treatment Main study 4-week recovery period condition Males [n] Females[n] Males [n] Females [n] Vehicle 3 3 2 2 ASO_(low) 3 3 / / ASO_(high) 33 2 2

The results of the present study demonstrate a chronicintracerebroventricular ASO administration to be a non-toxic and safemedication for the treatment of a variety of different diseases.

Example 26: Determination of the Stability and the Biological Activityof an Antisense-Oligonucleotide in Different Vehicle Solutions

To investigate, whether there are any interaction effects of theantisense-oligonucleotides (Seq. ID No. 218b, Seq. ID No. 218c) and theinfusion solution, a 29-day pre-experiment was performed. Therefore, thetwo ASOs were reconstituted in different endotoxin-free vehiclesolutions (PBS, water for injection [WFI], 0.9% NaCl) and stored atdifferent conditions, respectively. Samples were collected every singleweek and were analyzed for pH-value, ASO stability, content, andintegrity by AEX-HPLC. Any change in efficacy conditions were tested byproving the potency of TGF-R_(II) mRNA knockdown in cell-culture assaywith each sample, respectively.

Description of Method:

The lyophilized ASOs were diluted with the respective vehicle solution(Water for injection, 0.9% NaCl, PBS) under sterile conditions (laminarflow, BIOWIZARD Golden GL-170 Ergoscience®, S1 conditions). The 1.5 mlEppendorf Cups were labeled and filled with 100 μl (AEX-HPLC) or 250 μl(target knock down) of the respective ASO solution (all steps underlaminar flow, BIOWIZARD Golden GL-1 70 Ergoscience®, S1 conditions, seepipetting/labeling scheme table 81). In the next step, all samples werestored at their respective storing conditions and samples were collectedevery single week (see sampling scheme table 82) and stored at −80° C.until analyzation.

TABLE 81 Labeling scheme for the ASO-vehicle-stability study. Thelabeling scheme was performed for Seq. ID No. 218b and Seq. ID No. 218c(each 10 μM and 0.24 mM) and for all three vehicles WFI, 0.9% NaCl, andPBS (=>12 different schemes). Day Vehicle (WFI, 0.9% NaCL Label or PBS)Condition 0 6 12 ASO X Baseline ASO [10 μM] [10 μM] X_Baseline ASO X−20° C. ASO [10 μM] ASO [10 μM] [10 μM] X_−20° C._Day 6 X_−20° C._Day 12ASO X +4° C. ASO [10 μM] ASO [10 μM] [10 μM] X_+4° C._Day 6 X_+4° C._Day 12 ASO X +20° C. ASO [10 μM] ASO [10 μM] [10 μM] X_+20° C. _Day 6X_+20° C. _Day 12 ASO X +37° C. ASO [10 μM] ASO [10 μM] [10 μM] X_+37°C._Day 6 X_+37° C. _Day 12 ASO X +40° C. ASO [10 μM] ASO [10 μM] [10 μM]X_40° C._Day 6 X_40° C. _Day 12 ASO X pH value ASO [10 μM] [10 μM] X_pHvalue_Day 0 ASO X Baseline ASO [0.24 mM] [0.24 mM] X_Baseline ASO X −20°C. ASO [0.24 mM] ASO [0.24 mM] [0.24 mM] X_−20° C._Day 6 X_−20° C._Day12 ASO X +4° C. ASO [0.24 mM] ASO [0.24 mM] [0.24 mM] X_+4° C._Day 6X_+4° C. _Day 12 ASO X +20° C. ASO [0.24 mM] ASO [0.24 mM] [0.24 mM]X_+20° C. _Day 6 X_+20° C. _Day 12 ASO X +37° C. ASO [0.24 mM] ASO [0.24mM] [0.24 mM] X_+37° C._Day 6 X_+37° C. _Day 12 ASO X +40° C. ASO [0.24mM] ASO [0.24 mM] [0.24 mM] X_40° C._Day 6 X_40° C. _Day 12 ASO X pHvalue ASO [0.24 mM] [0.24 mM] X_pH value_Day 0 Day Label 18 24 29 ASO[10 μM] ASO ASO [10 μM] ASO [10 μM] ASO [10 μM] [10 μM] X_−20° C._Day 18X_−20° C._Day 24 X_−20° C._Day 29 ASO ASO [10 μM] ASO [10 μM] ASO [10μM] [10 μM] X_+4° C. _Day 18 X_+4° C. _Day 24 X_+4° C. _Day 29 ASO ASO[10 μM] ASO [10 μM] ASO [10 μM] [10 μM] X_+20° C. _Day 18 X_+20° C. _Day24 X_+20° C. _Day 29 ASO ASO [10 μM] ASO [10 μM] ASO [10 μM] [10 μM]X_+37°_Day 18 X_+37°_Day 24 X_+37° C. _Day 29 ASO ASO [10 μM] ASO [10μM] ASO [10 μM] [10 μM] X_40° C. _Day 18 X_40° C. _Day 24 X_40° C. _Day29 ASO ASO [10 μM] [10 μM] X_pH value_Day 29 ASO [0.24 mM] ASO ASO [0.24mM] ASO [0.24 mM] ASO [0.24 mM] [0.24 mM] X_−20° C._Day 18 X_−20° C._Day24 X_−20° C._Day 29 ASO ASO [0.24 mM] ASO [0.24 mM] ASO [0.24 mM] [0.24mM] X_+4° C. _Day 18 X_+4° C. _Day 24 X_+4° C. _Day 29 ASO ASO [0.24 mM]ASO [0.24 mM] ASO [0.24 mM] [0.24 mM] X_+20° C. _Day 18 X_+20° C. _Day24 X_+20° C. _Day 29 ASO ASO [0.24 mM] ASO [0.24 mM] ASO [0.24 mM] [0.24mM] X_+37°_Day 18 X_+37°_Day 24 X_+37° C. _Day 29 ASO ASO [0.24 mM] ASO[0.24 mM] ASO [0.24 mM] [0.24 mM] X_40° C. _Day 18 X_40° C. _Day 24X_40° C. _Day 29 ASO ASO [0.24 mM] [0.24 mM] X_pH_value_Day 29

TABLE 82 Collection scheme for the ASO-vehicle-stability study. Thecollection scheme was performed for Seq. ID No. 218b and Seq. ID No.218c (each 10 μM and 0.24 mM) and for all three vehicles WFI, 0.9% NaCl,and PBS (=>12 different schemes). Sample Day Condition 0 6 12 18 24 29Baseline X −20° C. X X X X X  +4° C. X X X X X +20° C. X X X X X +37° C.X X X X X +40° C. X X X X X pH value X X

Since there were no effects of any of the vehicle solutions onstability, content, and integrity of Seq. ID No. 218b and Seq. ID No.218c, 0.9% NaCl was used for the ASO-in use-stability experiment.

Example 27: Determination of the in-Use Stability and the BiologicalActivity of Inventive Antisense-Oligonucleotides (ASOs) in VehicleSolution

To investigate, whether there are any interaction effects of theantisense oligonucleotides (ASO) (Seq. ID No. 218b, Seq. ID No. 218c)and a gas pressure pump or a catheter, a 29-day pre-experiment wasperformed. Therefore, the two ASOs were reconstituted in 0.9% NaCl andthe pump and the catheter were filled according to manufacturer'sdescription. Samples were collected every single week and were analyzedfor pH-value, microbiology, and oligo stability, content, and integrityby AEX-HPLC. Any change in efficacy conditions were also tested byproofing the potency to knockdown TGF-R_(II) mRNA in cell-culture assaywith every sample, respectively.

Description of Method:

The lyophilized ASOs were diluted with 0.9% NaCl under sterileconditions (laminar flow, BIOWIZARD Golden GL-170 Ergoscience®, S1conditions). The 5 ml Eppendorf Cups were labeled according to thelabeling scheme (see table 83) under sterile conditions (laminar flow,BIOWIZARD Golden GL-170 Ergoscience, S1 conditions). The two gaspressure pumps (Tricumed Model IP-2000 V®) and the catheter (spinalcatheter set 4000) were filled according to manufacturer's descriptionwith the respective ASO solution (all steps under laminar flow,BIOWIZARD Golden GL-170 Ergoscience®, S1 conditions, seepipetting/labeling scheme table 83). In the next step, the pumpconnected to the catheter which was connected to the lid of a 5 mlEppendorf Cup and the remaining Cups were stored in a storage box withall openings being closed with Parafilm®, to avoid any contamination.Every single week the samples were collected, stored at −80° C. untilanalysis and the catheter connected to the lid of a 5 ml Eppendorf Cupwas transferred to the following Cup to continue the sampling procedure.In addition, one sample was taken directly from the pump via the bolusport and stored at −80° C. On the last day, an additional sample formicrobiological analysis was collected.

TABLE 83 Labeling scheme for the ASO in-use-stability study. Thelabeling scheme was performed for Seq. ID No. 218b and Seq. ID No. 218c(each 0.24 mM). Sample Day Oligo Cup Condition 0 6 12 18 24 29 Seq. IDNo. 5 ml PS Seq. ID No. Seq. ID No. Seq. ID No. Seq. ID No. Seq. ID No.Seq. ID No. 218b 218b 218b _+37° C. 218b _+37° C. 218b _+37° C. 218b_+37° C. 218b _+37° C. [0.24 mM] [10 μM] PS_Day 6 PS_Day 12 PS_Day 18PS_Day 24 PS_Day 29 Baseline Seq. ID No. 5 ml AS Seq. ID No. Seq. ID No.Seq. ID No. Seq. ID No. Seq. ID No. 218b 218b _+37° C. 218b _+37° C.218b _+37° C. 218b _+37° C. 218b _+37° C. [0.24 mM] AS_Day 6 AS_Day 12AS_Day 18 AS_Day 24 AS_Day 29 Seq. ID No. 5 ml MB Seq. ID No. 218b 218b_+37° C. [0.24 mM] MB_Day 29 Seq. ID No. 5 ml pH value Seq. ID No. Seq.ID No. 218b 218b 218b [0.24 mM] pH value pH value Day 0 Day 29 Seq. IDNo. 5 ml PS Seq. ID No. Seq. ID No. Seq. ID No. Seq. ID No. Seq. ID No.Seq. ID No. 218c 218c 218c _+37° C. 218c _+37° C. 218c _+37° C. 218c_+37° C. 218c _+37° C. [0.24 mM] Baseline PS_Day 6 PS_Day 12 PS_Day 18PS_Day 24 PS_Day 29 Seq. ID No. 5 ml AS Seq. ID No. Seq. ID No. Seq. IDNo. Seq. ID No. Seq. ID No. 218c 218c _+37° C. 218c _+37° C. 218c _+37°C. 218c _+37° C. 218c _+37° C. [0.24 mM] AS_Day 6 AS_Day 12 AS_Day 18AS_Day 24 AS_Day 29 Seq. ID No. 5 ml MB Seq. ID No. 218c 218c _+37° C.[0.24 mM] MB_Day 29 Seq. ID No. 5 ml pH value Seq. ID No. Seq. ID No.218c 218c 218c [0.24 mM] pH value pH value Day 0 Day 29 PS: (PumpSample:sample directly from the catheter), AS: (AdditionalSample: sampledirectly from the reservoir inside the pump via bolus port, MB:(MicroBiology: 500 μM from PS and AS)

TABLE 84 Collection scheme for the ASO in-use-stability study. Thecollection scheme was performed for Seq. ID No. 218b and Seq. ID No.218c (0.24 mM). Sample Day Cup Condition 0 6 12 18 24 29 5 ml Baseline X5 ml PS X X X X X 5 ml AS X X X X X 5 ml MB X 5 ml pH value X X PS:(PumpSample: sample directly from the catheter), AS: (AdditionalSample:sample directly from the reservoir inside the pump via bolus port, MB:(MicroBiology: 500 μM from PS and AS)

Since there were no effects of the pump and the catheter on thestability, content, and integrity of Seq. ID No. 218b and Seq. ID No.218c, and there were also no noticeable microbiological problems, thisapplication paradigm represents the optimal technique for theintrathecal and intracerebroventricular administration in Cynomolgusmonkeys and humans.

Chemical Synthesis Abbreviations

-   Pybop:    (Benzotriazol-1-yl-oxy)tripyrrolidinophosphonium-hexafluorophosphat-   DCM: Dichloromethane-   DMF: Dimethylformamide-   DMAP: 4-Dimethylaminopyridine-   DMT: 4,4′-dimethoxytrityl-   LCAA: Long Chain Alkyl Amino-   TRIS: Tris(hydroxymethyl)-aminomethan-   TRIS-HCl: Tris(hydroxymethyl)-aminomethan hydrochloride-   DEPC: Diethyl dicarbonate

Gapmer Antisense-Oligonucleotide Synthesis and Purification

The antisense-oligonucleotides in form of gapmers were assembled on anABI 3900 or on an ABI 394 synthesizer, or on an Expedite™ (AppliedBiosystems) according to the phosphoramidite oligomerization chemistry.On the AB13900, the solid support was polystyrene loaded with UnySupport(purchased from Glen Research, Sterling, Va., USA) to give a synthesisscale of 0.2 μmol. On the ABI 394 the solid support was 500 A controlledpore glass (CPG) loaded with Unylinker™ purchased from Chemgenes(Wilmington, Mass., USA) to give a 3 μmol synthesis scale.

Ancillary synthesis reagents such as “Deblock”, “Oxidizer”, “CapA” and“CapB” as well as DNA phosphoramidites were obtained from SAFC Proligo(Hamburg, Germany).

Specifically,5′-O-(4,4′-dimethoxytrityl)-2′-O,3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite monomers of deoxy thymidine (dT),4-N-benzoyl-2′-deoxy-cytidine (dC^(Bz)), 6-N-benzoyl-2′-deoxy-adenosine(dA^(Bz)) and 2-N-isobutyryl-2′-deoxy-guanosine (dG^(iBu)) were used asDNA building-units.5′-O-DMT-2′-O,4′-C-methylene-N²-dimethylformamidine-guanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (LNA-G^(DMF)),5′-O-DMT-2′-O,4′-C-methylene-thymidine3′-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite (LNA-Tb),5′-O-DMT-2′-O,4′-C-methylene-N⁶-benzoyladenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(LNA-A^(Bz)),5′-O-DMT-2′-O—,4′-C-methylene-5-methyl-N⁴-benzoylcytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(LNA-C*^(Bz)) were used as LNA-building-units. The LNA phosphoramiditeswere purchased from Exiqon (Vebaek, Denmark).

As shown by the examples of the LNAs in table 85, the phosphoramiditeswere dissolved in dry acetonitrile to give 0.07 M-oligonucleotide exceptLNA-C*^(Bz) which was dissolved in a mixture of THF/acetonitrile (25/75(v/v)).

TABLE 85 To obtain Molecular a 0.07M weight solution g/mole CAS No.Solvent 100 mg LNA-A^(Bz) 885.9 [206055-79-0] Anhydrous 1.6 mlAcetonitrile LNA-C*^(Bz) 875.9 [206055-82-5] THF/Acetonitrile 1.6 ml25/75 (v/v) LNA-G^(DMF) 852.9 [709641-79-2] Anhydrous 1.7 mlAcetonitrile LNA-T 772.8 [206055-75-6] Anhydrous 1.8 ml Acetonitrile

The β-D-thio-LNAs5′-O-DMT-2′-deoxy-2′-mercapto-2′-S,4′-C-methylene-N⁶-benzoyladenosine-3′-[(2-cyanoethyl-N,N-diisopropyl)]-phosphoramidite,5′-O-DMT-2′-deoxy-2′-mercapto-2′-S,4′-C-methylene-5-methyl-N⁴-benzoylcytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidites,5′-O-DMT-2′-deoxy-2′-mercapto-2′-S,4′-C-methylene-N²-dimethyformamidineguanosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,5′-O-DMT-2′-deoxy-2′-mercapto-2′-S,4′-C-methylene-thymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and5′-O-DMT-2′-deoxy-2′-amino-2′-N,4′-C-methylene-N⁶-benzoyladenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramiditeswere synthesized as described in J. Org. Chem. 1998, 63, 6078-6079.

The synthesis of the β-D-amino-LNA5′-O-DMT-2′-deoxy-2′-amino-2′-N,4′-C-methylene-5-methyl-N⁴-benzoylcytidine-3′-[(2-cyanoethyl-N,N-diisopropyl)]-phosphoramidites,5′-O-DMT-2′-deoxy-2′-amino-2′-N,4′-C-methylene-N²-dimethyformamidineguanosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,5′-O-DMT-2′-deoxy-2′-amino-2′-N,4′-C-methylene-thymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,5′-O-DMT-2′-deoxy-2′-methylamino-2′-N,4′-C-methylene-N⁶-benzoyladenosine-3′-[(2-cyanoethyl-N,N-diisopropyl)]-phosphoramidite,and5′-O-DMT-2′-deoxy-2′-methylamino-2′-N,4′-C-methylene-5-methyl-N⁴-benzoylcytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidites,5′-O-DMT-2′-deoxy-2′-methylamino-2′-N,4′-C-methylene-N²-dimethyformamidineguanosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite5′-O-DMT-2′-deoxy-2′-methylamino-2′-N,4′-C-methylene-thymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramiditewere carry out according to the literature procedure (J. Org Chem. 1998,63, 6078-6079).

Theα-L-oxy-LNAs_α-L-5′-O-DMT-2′-O,4′-C-methylene-N⁶-benzoyladenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite,α-L-5′-O-DMT-2′-O—,4′-C-methylene-5-methyl-N⁴-benzoylcytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite,α-L-5′-O-DMT-2′-O,4′-C-methylene-N²-dimethyformamidineguanosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,andα-L-5′-O-DMT-2′-O,4′-C-methylene-thymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramiditewere performed similar to the procedures described in the literature (J.Am. Chem. Soc. 2002, 124, 2164-2176; Angew. Chem. Int. Ed. 200, 39,1656-1659).

The (β-benzoylmercapto)ethyl)pyrrolidinolthiophosphoramidites for thesynthesis of the oligonucleotide with phosphorothioate backbone wereprepared in analogy to the protocol reported by Caruthers (J. Org. Chem.1996, 61, 4272-4281). The “phosphoramidites-C₃”(3-(4,4′-Dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramiditeand the “3′-Spacer C3 CPG”(1-Dimethoxytrityloxy-propanediol-3-succinoyl)-long chain alkylamino-CPGwere purchased from Glen Research.

General Procedure

Preparation of the LNA-Solid Support:

1) Preparation of the LNA Succinyl Hemiester (WO2007/112754)

-   -   5′-O-DMT-3′-hydroxy-nucleoside monomer, succinic anhydride (1.2        eq.) and DMAP (1.2 eq.) were dissolved in 35 ml dichloromethane        (DCM). The reaction was stirred at room temperature overnight.        After extractions with NaH₂PO₄ 0.1 M pH 5.5 (2×) and brine (1×),        the organic layer was further dried with anhydrous NaSO₄        filtered and evaporated. The hemiester derivative was obtained        in 95% yield and was used without any further purification.

2) Preparation of the LNA-Support (WO2007/112754)

-   -   The above prepared hemiester derivative (90 μmol) was dissolved        in a minimum amount of DMF, DIEA and pyBOP (90 μmol) were added        and mixed together for 1 min. This pre-activated mixture was        combined with LCAA-CPG (500 Å, 80-120 mesh size, 300 mg) in a        manual synthesizer and stirred. After 1.5 hours at room        temperature, the support was filtered off and washed with DMF,        DCM and MeOH. After drying, the loading was determined to be 57        μmol/g (see Tom Brown, Dorcas J. S. Brown. Modern machine-aided        methods of oligodeoxyribonucleotide synthesis. In: F. Eckstein,        editor. Oligonucleotides and 35 Analogues A Practical Approach.        Oxford: IRL Press, 1991: 13-14).

Elongation of the Oligonucleotide (Couplinq)

5-ethylthio-1H-tetrazole (ETT) as activator (0.5 M in acetonitrile) wasemployed for the coupling of the phosphoramidites. Instead of ETT otherreagents such as 4,5-dicyanoimidazole (DCI) as described inWO2007/112754, 5-benzylthio-1H-tetrazole or saccharin-1-methylimidazolcan be used as activator. 0.25 M DCI in acetonitrile was used for thecoupling with LNA.

Capping

10% acetic anhydride (Ac₂O) in THF (HPLC grade) and 10% N-methylimidazol(NMI) in THF/pyridine (8:1) (HPLC grade) were added and allowed toreact.

Oxidation

Phosphorous(III) to Phosphorous(V) is normally done with e.g.iodine/THF/pyridine/H₂O using 0.02 M iodine in THF/Pyridine/H₂Opurchased from Glen Research or 0.5 M1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO) in anhydrous acetonitrilefrom Glen Research.

In the case that a phosphorthioate internucleoside linkage is prepared,a thiolation step is performed using a 0.05 M solution of3-((dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT,obtained from Chemgenes (Wilmington, Mass., USA)) in anhydrousacetonitrile/pyridine (1:1 v/v). In case LNAs are used, the thiolationwas carried out usind 0.2 M 3,H-1,2-benzothiole-3-one 1,1-dioxide(Beaucage reagent) in anhydrous acetonitrile.

In general, the thiolation can also be carried out by using xanthanechloride (0.01 M in acetonitrile/pyridine 10%) as described inWO2007/112754.

Alternative, other reagents for the thiolation step such as xanthanehydride (5-imino-(1,2,4)dithiazolidine-3-thione), phenylacetyl disulfide(PADS) can be applied.

In the case that a phosphordithioate was synthesized, the resultingthiophosphite triester was oxidized to the phosphorothiotriester byaddition of 0.05 M DDTT(3-((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione) inpyridine/acetonitrile (4:1 v/v).

Cleavage from the Solid Support and Deprotection

At the end of the solid phase synthesis, the antisense-oligonucleotidecan either be cleaved “DMT-on” or “DMT-off”. “DMT off” means that thefinal 5′-O-(4,4′-dimethoxytrityl) group was removed on the synthesizerusing the “Deblock” reagent and DMT-on means that the group is presentwhile the oligonucleotide is cleaved from the solid support. The DMTgroups were removed with trichloroacetic acid.

“Dmt-Off”

Upon completion of the solid phase synthesis antisense-oligonucleotideswere treated with a 20% diethylamine solution in acetonitrile (BiosolveBV, Valkenswaard, The Netherlands) for 20 min. to remove the cyanoethylprotecting groups on the phosphate backbone. Subsequently, theantisense-oligonucleotides were cleaved from the solid support anddeprotected using 1 to 5 mL concentrated aqueous ammonia (obtained fromSigma Aldrich) for 16 hours at 55° C. The solid support was separatedfrom the antisense-oligonucleotides by filtration or centrifugation.

If the oligonucleotides contain phosphorodithioate triester, thethiol-groups were deprotected with thiophenol:triethylamine:dioxane,1:1:2, v/v/v for 24 h, then the oligonucleotides were cleaved from thesolid support using aqueous ammonia for 1-2 hours at room temperature,and further deprotected for 4 hours at 65° C.

“DMT-on”

The oligonucleotides were cleaved from the solid support using aqueousammonia for 1-2 hours at room temperature, and further deprotected for 4hours at 65° C. The oligonucleotides were purified by reverse phase HPLC(RP-HPLC), and then the DMT-group is removed with trichloroacetic acid.

If the oligonucleotides contain phosphorodithioate triester, thecleavage from the solid-support and the deprotection of the thiol-groupwere performed by the addition of 850 μl ammonia in concentrated ethanol(ammonia/ethanol 3:1 v/v) at 55° C. for 15-16h.

Terminal Groups

Terminal groups at the 5′-end of the antisense oligonucleotide

The solid supported oligonucleotide was treated with 3% trichloroaceticacid in dichloromethane (w/v) to completely remove the 5′-DMT protectiongroup. Further, the compound was converted with an appropriate terminalgroup with cyanoethyl-N,N-diisopropyl)phosphoramidite-moiety. After theoxidation of the phosphorus (III) to phosphorus(V), the deprotection,detachment from the solid support and deprotection sequence wereperformed as described above.

Purification

Next, the crude antisense-oligonucleotides were purified byanion-exchange high-performance liquid chromatography (HPLC) on an AKTAExplorer System (GE Healthcare, Freiburg, Germany) and a column packedwith Source Q15 (GE Helthcare). Buffer A was 10 mM sodium perchlorate,20 mM Tris, 1 mM EDTA, pH 7.4 and contained 20% acetonitrile and bufferB was the same as buffer A with the exception of 500 mM sodiumperchlorate. A gradient of 15% B to 55% B within 32 column volumes (CV)was employed. UV traces at 280 nm were recorded. Appropriate fractionswere pooled and precipitated with 3 M NaOAc, pH=5.2 and 70% ethanol.Finally, the pellet was washed with 70% ethanol. Analytics

Identity of the antisense-oligonucleotides was confirmed by electrosprayionization mass spectrometry (ESI-MS) and purity was by analyticalOligoPro Capillary Electrophoresis (CE).

The purification of the dithioate was performed on an AmershamBiosciences P920 FPLC instrument fitted with a Mono Q 10/100 GL column.The buffers were prepared with DEPC-treated water, and theircompositions were as follows: Buffer A: 25 mM Tris-HCl, 1 mM EDTA, pH8.0; Buffer B: 25 mM Tris-HCl, 1 mM EDTA, 1 M NaCl, pH 8.0.

Example 28

Gb¹sTb¹sdAsdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsAb¹sGb¹sC*b¹ (Seq. ID No. 209y)

5′-O-DMT-2′-O,4′-C-methylene-5-methyl-N⁴-benzoxylcytidine-3′-O-succinoyl-linked LCAA CPG (0.2 μmol) was treated with 1400 μl 3% trichloroaceticacid in dichloromethane for 60 s to completely remove the 5′-DMTprotection group. After several washes with a total amount of 800 μlacetonitrile, the coupling reaction was carried out with 80 μl5′-O-DMT-2′-O,4′-C-methylene-N²-dimethyformamidineguanosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-O,4′-C-methylene-N⁶-benzoyladenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidites(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N²-isobutyryl-2′-deoxyguanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidites(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N²-isobutyryl-2′-deoxyguanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidites(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N²-isobutyryl-2′-deoxyguanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidites(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁶-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-deoxythymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-deoxythymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-deoxythymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N²-isobutyryl-2′-deoxyguanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidites(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-deoxythymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N²-isobutyryl-2′-deoxyguanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁶-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidites(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl 5′-O-DMT-2′-O,4′-C-methylenethymidine 3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (0.07 M)and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction wasallowed to take place for 250 sec., and excess reagents were flashed outwith 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were insertedto the column for 180 s The system was flushed with 320 μl acetonitrile.For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) were added andallowed to react for 45 sec. At the end of this cycle, the system waswashed with 480 μl acetonitrile. The compound was treated with 1400 μl3% trichloroacetic acid in dichloromethane for 60 s to completely removethe 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-O,4′-C-methylene-N²-dimethyformamidineguanosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

Upon completion of the solid phase synthesis, theantisense-oligonucleotides were treated with a 20% diethylamine solutionin acetonitrile (Biosolve BV, Valkenswaard, The Netherlands) for 20 min.to remove the cyanoethyl protecting groups on the phosphate backbone.

Subsequently, the antisense-oligonucleotides were cleaved from the solidsupport and further deprotected using 5 mL concentrated aqueous ammoniafor 16 hours at 55° C. The solid support was separated from theantisense-oligonucleotides by filtration or centrifugation.

Next, the crude antisense-oligonucleotides were purified byanion-exchange high-performance liquid chromatography (HPLC) on an AKTAExplorer System (GE Healthcare, Freiburg, Germany) and a column packedwith Source Q15 (GE Helthcare). Buffer A was 10 mM sodium perchlorate,20 mM Tris, 1 mM EDTA, pH 7.4 and contained 20% acetonitrile and bufferB was the same as buffer A with the exception of 500 mM sodiumperchlorate. A gradient of 15% B to 55% B within 32 column volumes (CV)was employed. UV traces at 280 nm were recorded. Appropriate fractionswere pooled and precipitated with 3 M NaOAc, pH=5.2 and 70% ethanol.Finally, the pellet was washed with 70% ethanol. The antisenseoligonucleotide was received with a purity of 93.7%. ESI-MS:experimental: 5387.3 Da; calculated: 5387.80 Da.

Example 29

(Seq. ID No. 209u) Gb¹Tb¹dAdGdTdGdTdTdTdAdGdGdGAb¹Gb¹C*b¹

The LNA was bound to CPG according to the general procedure. Thecoupling reaction and capping step were also carried out as described inexample 28. After the capping step, the system was flushed out with 800μl acetonitrile, and 400 μl of 0.02 M Iodine in THF/pyridine/H₂O wereinserted to the column for 45 s. The system was flushed after theoxidation step with 24 μl acetonitrile. After purification, theantisense oligonucleotide was received with a purity of 95.3%. ESI-Ms:experimental: 5146.80 Da; calculated: 5146.4 Da.

Example 30

(Seq. ID No. 209v) /5SpC3s/Gb¹sTb¹sdAsdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsAb¹sGb¹sC*b¹

The compound was synthesized according to the general procedure with theappropriate DNA and LNA building units as exemplified in example 28. Butwith the exception that after the last nucleotide has been coupled tothe oligonucleotide and the subsequent oxidation and capping steps werecarried out, 80 μl of phosphoramidite-C₃ (0.07 M) and 236 μl DCI inacetonitrile (0.25 M) were added. The coupling was allowed to take placefor 250 sec., and excess reagents were flashed out with 800 μlacetonitrile. The subsequent steps were performed as described inexample 28. After purification, the antisense oligonucleotide wasreceived with a purity of 97.4%. HRMS (ESI): experimental: 5540.70 Da;calculated: 5541.4 Da.

Example 31

(Seq. ID No. 209w) Gb¹sTb¹sdAsdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsAb¹sGb¹sC*b¹/3SpC3s/

3′-Spacer C3 CPG (0.2 μmol) was treated with 1400 μl 3% trichloroaceticacid in dichloromethane for 60 s to completely remove the 5′-DMTprotection group. After several washes with a total amount of 800 μlacetonitrile, the coupling reaction was carried out with 80 μl5′-O-DMT-2′-O—,4′-C-methylene-5-methyl-N⁴-benzoylcytidine-3′-[(2-cyanoethyl-N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group. The subsequent reactionswere performed as described in example 28. After purification, theantisense oligonucleotide was received with a purity of 92.7%. ESI-sMS:experimental: 5541.70 Da; calculated: 5541.4 Da.

Example 32

(Seq. ID No. 209an) Gb¹ssTb¹ssAb¹ssdGssdTssdGssdTssdTssdTssdA*ssdGssdGssdGssAb¹ssGb¹ssC*b¹

5′-O-DMT-2′-O,4′-C-methylene-5-methyl-N⁴-benzoxylcytidine-3′-O-succinoyl-linked LCAA CPG (0.2 μmol) was treated with 1400 μl 3% trichloroaceticacid in dichloromethane for 60 s to completely remove the 5′-DMTprotection group. After several washes with a total amount of 800 μlacetonitrile, the coupling reaction was carried out with 38 μl5′-O-DMT-2′-O,4′-C-methylene-N²-dimethyformamidineguanosine-3′-[(β-benzoylmercapto)ethyl]pyrrolidinolthiophosphoramidite(0.15 M) in 10% dichloromethane (v/V) in acetonitrile and 236 μl DCI inacetonitrile (0.25 M). The coupling reaction was allowed to take placefor 250 sec., and excess reagents were flashed out with 800 μlacetonitrile, and 900 μl of DDTT (0.05 M in pyridine/acetonitrile 4:1v/v) were inserted to the column for 240 s The system was flushed with320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride inTHF (1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1)were added and allowed to react for 45 sec. At the end of this cycle,the system was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 38 μl5′-O-DMT-2′-O,4′-C-methylene-N⁶-benzoyladenosine-3′-[(3-benzoylmercapto)ethyl]pyrrolidinolthiophosphoramidite(0.15 M) in 10% dichloromethane (v/v) in acetonitrile and 236 μl DCI inacetonitrile (0.25 M). The coupling reaction was allowed to take placefor 250 sec., and excess reagents were flashed out with 800 μlacetonitrile, and 900 μl of DDTT (0.05 M in pyridine/acetonitrile 4:1v/v) were inserted to the column for 240 s The system was flushed with320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride inTHF (1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1)were added and allowed to react for 45 sec. At the end of this cycle,the system was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The further elongation of the oligonucleotide was performed in the sameway.

Upon completion of the solid phase synthesis, theantisense-oligonucleotides were treated 850 μl ammonia in concentratedethanol (ammonia/ethanol 3:1 v/v) at 55° C. for 15-16h in order tocleave antisense-oligonucleotide from the solid-support and to deprotectthe thiol-group.

Next, the crude antisense-oligonucleotide was purified by anion-exchangechromatogtraophy using a Mono Q 10/100 GL column. The buffers wereprepared with DEPC-treated water, and their compositions were asfollows: Buffer A: 25 mM Tris-HCl, 1 mM EDTA, pH 8.0; Buffer B: 25 mMTris-HCl, 1 mM EDTA, 1 M NaCl, pH 8.0.

Example 33

(Seq. ID No. 209az) Gb¹sTb¹sAb¹sdGsdTsdGsdTsdTsdTsdAsdGsdGsGbsAb¹sGb¹sC*b¹

The compound was synthesized according to the general procedure with theappropriate DNA and LNA building units as exemplified in example 28.After purification, the antisense oligonucleotide was received with apurity of 90.5%. ESI-MS: experimental: 5442.9 Da; calculated: 5443.3 Da.

Example 34

(Seq. ID No. 209ba) Gb¹sTb¹sAb¹sGb¹sdTsdGsdTsdTsdTsdAsdGsdGsGb¹sAb¹sGb¹sC*b¹

The compound was synthesized according to the general procedure with theappropriate DNA and LNA building units as exemplified in example 28.After purification, the antisense oligonucleotide was received with apurity of 89.4%. ESI-MS: experimental: 5469.9 Da; calculated: 5471.3 Da.

(Seq. ID No. 209bb) Gb¹sTb¹sAb¹sdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsdAsGb¹sC*b¹

Example 35

The compound was synthesized according to the general procedure with theappropriate DNA and LNA building units as exemplified in example 28.After purification, the antisense oligonucleotide was received with apurity of 88.4%. ESI-MS: experimental: 5386.5 Da; calculated: 5387.3 Da.

Example 36

(Seq. ID No. 209s) Gb¹Tb¹dAsdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsAb¹Gb¹C*b¹

The compound was synthesized according to the procedure as described inexample 28 and example 29 with the appropriate DNA, DNA-derivatives andthe LNA building units. After purification, the antisenseoligonucleotide was received with a purity of 96.8%. ESI-MS:experimental: 5323.30 Da; calculated: 5323.0 Da.

Example 37

(Seq. ID No. 209t) Gb¹sTb¹sdA*sdGsdTsdGsdTsdTsdTsdA*sdGsdGsdGsAb¹sGb¹sC*b¹

The compound was synthesized according to the general procedure and asdescribed in example 28 with the appropriate DNA and LNA building units.After purification, the antisense oligonucleotide was received with apurity of 91.4%. ESI-MS: experimental: 5416.30 Da; calculated: 5417.3Da.

Example 38

(Seq. ID No. 209x) /5SpC3s/Gb¹sTb¹sdAsdGsdTsdGsdTsdTsdTsdAsdGsdGsdGsAb¹sGb¹sC*b¹/3SpC3s/

The compound was synthesized according to the general procedure with theappropriate DNA and LNA building units as exemplified in example 28,example 30 and example 31. After purification, the antisenseoligonucleotide was received with a purity of 95.1%. ESI-MS:experimental: 5696.30 Da; calculated: 5695.5 Da.

Examples 39-132

The other oligonucleotides of Table 6 were synthesized according to thegeneral procedure and as shown in the examples. The preparation of theantisense-oligonucleotide including β-D-thio-LNA, α-L-oxy-LNA,β-D-(NH)-LNA, or β-D-(NCH₃)-LNA units were performed in the same way asthe antisense-oligonucleotides containing β-D-oxy-LNA units.

Example 133

(Seq. ID No. 210q) Gb¹sC*b¹sTb¹sAb¹sdTsdTsdTsdGsdGsdTsdAsdGsdTsGb¹sTb¹sTb¹

5′-O-DMT-2′-O,4′-C-methylene thymidine-3′-O-succinoyl- linked LCAA CPG(0.2 μmol) was treated with 1400 μl 3% trichloroacetic acid indichloromethane for 60 s to completely remove the 5′-DMT protectiongroup. After several washes with a total amount of 800 μl acetonitrile,the coupling reaction was carried out with 80 μl5′-O-DMT-2′-O,4′-C-methylene thymidine3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (0.07 M) and 236μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed totake place for 250 sec., and excess reagents were flashed out with 800μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to thecolumn for 180 s The system was flushed with 320 μl acetonitrile. Forthe capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μlN-methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed toreact for 45 sec. At the end of this cycle, the system was washed with480 μl acetonitrile. The compound was treated with 1400 μl 3%trichloroacetic acid in dichloromethane for 60 s to completely removethe 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-O,4′-C-methylene-N²-dimethyformamidineguanosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-deoxythymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N²-isobutyryl-2′-deoxyguanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidites(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁶-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-deoxythymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N²-isobutyryl-2′-deoxyguanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N²-isobutyryl-2′-deoxyguanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-deoxythymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-deoxythymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-deoxythymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-O,4′-C-methylene-N⁶-benzoyladenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl 5′-O-DMT-2′-O,4′-C-methylenethymidine 3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (0.07 M)and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction wasallowed to take place for 250 sec., and excess reagents were flashed outwith 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were insertedto the column for 180 s The system was flushed with 320 μl acetonitrile.For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) were added andallowed to react for 45 sec. At the end of this cycle, the system waswashed with 480 μl acetonitrile. The compound was treated with 1400 μl3% trichloroacetic acid in dichloromethane for 60 s to completely removethe 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-O—,4′-C-methylene-5-methyl-N⁴-benzoylcytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-O,4′-C-methylene-N²-dimethyformamidineguanosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

Upon completion of the solid phase synthesis antisense-oligonucleotideswere treated with a 20% diethylamine solution in acetonitrile (BiosolveBV, Valkenswaard, The Netherlands) for 20 min. to remove the cyanoethylprotecting groups on the phosphate backbone.

Subsequently, the antisense-oligonucleotides were cleaved from the solidsupport and further deprotected using 5 ml concentrated aqueous ammoniafor 16 hours at 55° C. The solid support was separated from theantisense-oligonucleotides by filtration or centrifugation.

Next, the crude antisense-oligonucleotides were purified byanion-exchange high-performance liquid chromatography (HPLC) on an AKTAExplorer System (GE Healthcare, Freiburg, Germany) and a column packedwith Source Q15 (GE Helthcare). Buffer A was 10 mM sodium perchlorate,20 mM Tris, 1 mM EDTA, pH 7.4 and contained 20% acetonitrile and bufferB was the same as buffer A with the exception of 500 mM sodiumperchlorate. A gradient of 15% B to 55% B within 32 column volumes (CV)was employed. UV traces at 280 nm were recorded. Appropriate fractionswere pooled and precipitated with 3 M NaOAc, pH=5.2 and 70% ethanol.Finally, the pellet was washed with 70% ethanol.

The antisense oligonucleotide was received with a purity of 87.1%.ESI-MS: experimental: 5384.30 Da; calculated: 5384.3 Da.

Example 134

(Seq. ID No. 210r) Gb ¹ C*b ¹ Tb ¹ Ab ¹dTdTdTdGdGdTdA*dGdTGb ¹ Tb ¹ Tb ¹

The LNA was bound to CPG according to the general procedure. Thecoupling reaction and capping step were also carried out as described inexample 133. After the capping step, the system was flushed out with 800μl acetonitrile, and 400 μl of 0.02 M Iodine in THF/pyridine/H₂O wereinserted to the column for 45 s. The system was flushed after theoxidation step with 24 μl acetonitrile. After purification, theantisense oligonucleotide was received with a purity of 95.3%.

Example 135

(Seq. ID No. 210v) /5SpC3s/Gb ¹ sC*b ¹ sTb ¹ sAb ¹sdTsdTsdTsdGsdGsdTsdAsdGs dTsGb ¹ sTb ¹ sTb ¹

The compound was synthesized according to the general procedure with theappropriate DNA and LNA building units as exemplified in example 133.But with the exception that after the last nucleotide has been coupledto the oligonucleotide and the subsequent oxidation and capping stepswere carried out, 80 μl of phosphoramidite-C₃ (0.07 M) and 236 μl DCI inacetonitrile (0.25 M) were added. The coupling was allowed to take placefor 250 sec., and excess reagents were flashed out with 800 μlacetonitrile. The subsequent steps were performed as described inexample 133. After purification, the antisense oligonucleotide wasreceived with a purity of 93.9%.

Example 136

(Seq. ID No. 210w) Gb ¹ sC*b ¹ sTb ¹ sAb ¹sdTsdTsdTsdGsdGsdTsdAsdGsdTsGb ¹ s Tb ¹ sTb ¹/3SpC3s/

3′-Spacer C3 CPG (0.2 μmol) was treated with 1400 μl 3% trichloroaceticacid in dichloromethane for 60 s to completely remove the 5′-DMTprotection group. After several washes with a total amount of 800 μlacetonitrile, the coupling reaction was carried out with 80 μl5′-O-DMT-2′-O,4′-C-methylene-thymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group. The subsequent reactionswere performed as described in example 133. After purification, theantisense oligonucleotide was received with a purity of 89.7%.

Example 137

(Seq. ID No. 210o) Gb¹C*b¹Tb¹Ab¹dTsdTsdTsdGsdGsdTsdAsdGsdTsGb¹Tb¹Tb¹

The compound was synthesized according to the general procedure with theappropriate DNA and LNA building units as exemplified in example 133 andexample 134. After purification, the antisense oligonucleotide wasreceived with a purity of 83.8%. ESI-MS: experimental: 5288.10 Da;calculated: 5287.9 Da.

Example 138

(Seq. ID No. 210p) Gb¹sC*b¹sTb¹sAb¹sdTsdTsdTdGsdGsdTsdA*sdGsdTsGb¹sTb¹sTb¹

The compound was synthesized according to the general procedure with theappropriate DNA and LNA building units as exemplified in example 133.After purification, the antisense oligonucleotide was received with apurity of 80.7%. ESI-MS: experimental: 5398.40 Da; calculated: 5399.3Da.

Example 139

(Seq. ID No. 210af) Gb ¹ ssC*b ¹ ssTb ¹ssdAssdTssdTssdTssdGssdGssdTssdA*ssd GssdTssGb ¹ ssTb ¹ ssTb ¹

5′-O-DMT-2′-O,4′-C-methylene-thymidine-3′-O-succinoyl- linked LCAA CPG(0.2 μmol) was treated with 1400 μl 3% trichloroacetic acid indichloromethane for 60 s to completely remove the 5′-DMT protectiongroup. After several washes with a total amount of 800 μl acetonitrile,the coupling reaction was carried out with 38 μl5′-O-DMT-2′-O,4′-C-methylene-thymidine-3′-[(β-benzoylmercapto)ethyl]pyrrolidinolthiophosphoramidite(0.15 M) in 10% dichloromethane (v/V) in acetonitrile and 236 μl DCI inacetonitrile (0.25 M). The coupling reaction was allowed to take placefor 250 sec., and excess reagents were flashed out with 800 μlacetonitrile, and 900 μl of DDTT (0.05 M in pyridine/acetonitrile 4:1v/v) were inserted to the column for 240 s The system was flushed with320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride inTHF (1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1)were added and allowed to react for 45 sec. At the end of this cycle,the system was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 38 μl5′-O-DMT-2′-O,4′-C-methylene-N²-dimethyformamidineguanosine-3′-[(β-benzoylmercapto)ethyl]pyrrolidinolthiophosphoramidite(0.15 M) in 10% dichloromethane (v/v) in acetonitrile and 236 μl DCI inacetonitrile (0.25 M). The coupling reaction was allowed to take placefor 250 sec., and excess reagents were flashed out with 800 μlacetonitrile, and 900 μl of DDTT (0.05 M in pyridine/acetonitrile 4:1v/v) were inserted to the column for 240 s The system was flushed with320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride inTHF (1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1)were added and allowed to react for 45 sec. At the end of this cycle,the system was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The further elongation of the oligonucleotide was performed in the sameway.

Upon completion of the solid phase synthesis, theantisense-oligonucleotides were treated 850 μl ammonia in concentratedethanol (ammonia/ethanol 3:1 v/v) at 55° C. for 15-16h in order tocleave antisense-oligonucleotide from the solid-support and to deprotectthe thiol-group.

Next, the crude antisense-oligonucleotide was purified by anion-exchangechromatogtraophy using a Mono Q 10/100 GL column. The buffers wereprepared with DEPC-treated water, and their compositions were asfollows: Buffer A: 25 mM Tris-HCl, 1 mM EDTA, pH 8.0; Buffer B: 25 mMTris-HCl, 1 mM EDTA, 1 M NaCl, pH 8.0.

Example 140-233

The other oligonucleotides of Table 7 were synthesized according to thegeneral procedure and as shown in the examples. The preparation of theantisense-oligonucleotide including β-D-thio-LNA, α-L-oxy-LNA,β-D-(NH)-LNA, or β-D-(NCH₃)-LNA units were performed in the same way asthe antisense-oligonucleotides containing β-D-oxy-LNA units.

Example 234

(Seq. ID No. 218b) C*b¹sAb¹sTb¹sdGsdAsdAsdTsdGsdGsdAsdCsdCsAb¹sGb¹sTb¹sAb¹

5′-O-DMT-2′-O,4′-C-methylene-N⁶-benzoyladenosine-3′-O-succinate

5′-O-DMT-2′-O,4′-C-methylene-N⁶-benzoyladenosine (500 mg, 0.73 mmol), 95mg succinic anhydride (0.95 mmol, 1.2 eq.) and 116 mg DMAP (0.95 mmol,1.2 eq.) were dissolved in 35 ml dichloromethane. The reaction wasstirred at room temperature overnight. The reaction solution was washed2 times with 10 ml NaH₂PO₄ (0.1 M, pH 5.5) and one time with 10 mlbrine. The organic phase was dried under anhydrous NaSO₄, filtered andconcentrated to dryness in vacuo. The hemiester derivative was obtainedin 95% yield and was used without further purification for the nextstep.

5′-O-DMT-2′-O,4′-C-methylene-N⁶-benzoyladenosine-3′-O-succinoyl- linkedLCAA CPG

70 mg hemiester derivative (90 μmol) was dissolved in 0.3 ml DMF, 11.6μl DIEA (90 μmol) and pyBOP (90 μmol) were added and mixed together for1 min. This mixture was combined with LCAA-CPG (500 A, 80-120 mesh size,300 mg) in a manual synthesizer and stirred for 1.5 hours at roomtemperature. The support was filtered off and washed with DMF, DCM andMeOH. After drying, the loading was determined to be 57 μmol/g.

Elongation

5′-O-DMT-2′-O,4′-C-methylene-N⁶-benzoxyladenosine-3′-O-succinoyl- linkedLCAA CPG (0.2 μmol) was treated with 1400 μl 3% trichloroacetic acid indichloromethane for 60 s to completely remove the 5′-DMT protectiongroup. After several washes with a total amount of 800 μl acetonitrile,the coupling reaction was carried out with 80 μl5′-O-DMT-2′-O,4′-C-methylene-thymidine3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (0.07 M) and 236μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed totake place for 250 sec., and excess reagents were flashed out with 800μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to thecolumn for 180 s The system was flushed with 320 μl acetonitrile. Forthe capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μlN-methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed toreact for 45 sec. At the end of this cycle, the system was washed with480 μl acetonitrile. The compound was treated with 1400 μl 3%trichloroacetic acid in dichloromethane for 60 s to completely removethe 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-O,4′-C-methylene-N²-dimethyformamidineguanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-O,4′-C-methylene-N⁶-benzoyladenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁴-benzoyl-2′-deoxycytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁴-benzoyl-2′-deoxycytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁶-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N²-isobutyryl-2′-deoxyguanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N²-isobutyryl-2′-deoxyguanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-deoxythymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁶-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁶-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N²-isobutyryl-2′-deoxyguanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl 5′-O-DMT-2′-O,4′-C-methylenethymidine 3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (0.07 M)and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction wasallowed to take place for 250 sec., and excess reagents were flashed outwith 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were insertedto the column for 180 s The system was flushed with 320 μl acetonitrile.For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) were added andallowed to react for 45 sec. At the end of this cycle, the system waswashed with 480 μl acetonitrile. The compound was treated with 1400 μl3% trichloroacetic acid in dichloromethane for 60 s to completely removethe 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-O,4′-C-methylene-N⁶-benzoyladenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-O—,4′-C-methylene-5-methyl-N⁴-benzoylcytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

Upon completion of the solid phase synthesis antisense-oligonucleotideswere treated with a 20% diethylamine solution in acetonitrile (BiosolveBV, Valkenswaard, The Netherlands) for 20 min. to remove the cyanoethylprotecting groups on the phosphate backbone.

Subsequently, the antisense-oligonucleotides were cleaved from the solidsupport and further deprotected using 5 mL concentrated aqueous ammoniafor 16 hours at 55° C. The solid support was separated from theantisense-oligonucleotides by filtration or centrifugation.

Next, the crude antisense-oligonucleotides were purified byanion-exchange high-performance liquid chromatography (HPLC) on an AKTAExplorer System (GE Healthcare, Freiburg, Germany) and a column packedwith Source Q15 (GE Helthcare). Buffer A was 10 mM sodium perchlorate,20 mM Tris, 1 mM EDTA, pH 7.4 and contained 20% acetonitrile and bufferB was the same as buffer A with the exception of 500 mM sodiumperchlorate. A gradient of 15% B to 55% B within 32 column volumes (CV)was employed. UV traces at 280 nm were recorded. Appropriate fractionswere pooled and precipitated with 3 M NaOAc, pH=5.2 and 70% ethanol.Finally, the pellet was washed with 70% ethanol. The antisenseoligonucleotide was received with a purity of 94.8%. ESI-MS:experimental: 5365.80 Da; calculated: 5365.30 Da.

Example 235

(Seq. ID No. 218r) C*b¹Ab¹Tb¹dGdAdAdTdGdGdAdCdCAb¹Gb¹Tb¹Ab¹

The LNA was bound to CPG according the general procedure. The couplingreaction and capping step were also carried out as described in example234. After the capping step, the system was flushed out with 800 μlacetonitrile, and 400 μl of 0.02 M Iodine in THF/pyridine/H₂O wereinserted to the column for 45 s. The system was flushed after theoxidation step with 24 μl acetonitrile. After purification, theantisense oligonucleotide was received with a purity of 97.8%. ESI-MS:experimental: 5125.10 Da; calculated: 5124.4 Da.

Example 236

(Seq. ID No. 218t) /5SpC3s/C*b ¹ sAb ¹ sTb ¹sdGsdAsdAsdTsdGsdGsdAsdCsdCs Ab ¹ sGb ¹ sTb ¹ sAb ¹

The compound was synthesized according to the general procedure with theappropriate DNA and LNA building units as exemplified in example 234.But with the exception that after the last nucleotide has been coupledto the oligonucleotide and the subsequent oxidation and capping stepswere carried out, 80 μl of phosphoramidite-C3 (0.07 M) and 236 μl DCI inacetonitrile (0.25 M) were added. The coupling was allowed to take placefor 250 sec., and excess reagents were flashed out with 800 μlacetonitrile. The subsequent steps were performed as described inexample 234. After purification, the antisense oligonucleotide wasreceived with a purity of 94.2%. ESI-MS: experimental: 5519.60 Da;calculated: 5519.4 Da.

Example 237

(Seq. ID No. 218u) C*b¹sAb¹sTb¹sdGsdAsdAsdTsdGsdGsdAsdCsdCsAb¹sGb¹sTb¹sAb¹s/3SpC3/

3′-Spacer C3 CPG (0.2 μmol) was treated with 1400 μl 3% trichloroaceticacid in dichloromethane for 60 s to completely remove the 5′-DMTprotection group. After several washes with a total amount of 800 μlacetonitrile, the coupling reaction was carried out with 80 μl5′-O-DMT-2′-O,4′-C-methylene-N⁶-benzoyladenosine-3′-[(2-cyanoethyl-N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group. The subsequent reactionswere performed as described in example 234. After purification, theantisense oligonucleotide was received with a purity of 94.3%. ESI-MS:experimental: 5519.10 Da; calculated: 5519.4 Da.

Example 238

(Seq. ID No. 218aa) C*b ¹ ssAb ¹ ssTb¹ssdGssdAssdAssdTssdGssdGssdAssdCssd CssAb ¹ ssGb ¹ ssTb ¹ ssAb ¹

5′-O-DMT-2′-O,4′-C-methylene-N⁶-benzoyladenosine-3′-O-succinoyl- linkedLCAA CPG (0.2 μmol) was treated with 1400 μl 3% trichloroacetic acid indichloromethane for 60 s to completely remove the 5′-DMT protectiongroup. After several washes with a total amount of 800 μl acetonitrile,the coupling reaction was carried out with 38 μl5′-O-DMT-2′-O,4′-C-methylene-thymidine-3′-[(β-benzoylmercapto)ethyl]pyrrolidinolthiophosphoramidite(0.15 M) in 10% dichloromethane (v/V) in acetonitrile and 236 μl DCI inacetonitrile (0.25 M). The coupling reaction was allowed to take placefor 250 sec., and excess reagents were flashed out with 800 μlacetonitrile, and 900 μl of DDTT (0.05 M in pyridine/acetonitrile 4:1v/v) were inserted to the column for 240 s The system was flushed with320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride inTHF (1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1)were added and allowed to react for 45 sec. At the end of this cycle,the system was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 38 μl5′-O-DMT-2′-O,4′-C-methylene-N²-dimethyformamidineguanosine-3′-[(β-benzoylmercapto)ethyl]pyrrolidinolthiophosphoramidite(0.15 M) in 10% dichloromethane (v/v) in acetonitrile and 236 μl DCI inacetonitrile (0.25 M). The coupling reaction was allowed to take placefor 250 sec., and excess reagents were flashed out with 800 μlacetonitrile, and 900 μl of DDTT (0.05 M in pyridine/acetonitrile 4:1v/v) were inserted to the column for 240 s The system was flushed with320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride inTHF (1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1)were added and allowed to react for 45 sec. At the end of this cycle,the system was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The further elongation of the oligonucleotide was performed in the sameway.

Upon completion of the solid phase synthesis, theantisense-oligonucleotides were treated 850 μl ammonia in concentratedethanol (ammonia/ethanol 3:1 v/v) at 55° C. for 15-16h in order tocleave antisense-oligonucleotide from the solid-support and to deprotectthe thiol-group.

Next, the crude antisense-oligonucleotide was purified by anion-exchangechromatogtraophy using a Mono Q 10/100 GL column. The buffers wereprepared with DEPC-treated water, and their compositions were asfollows: Buffer A: 25 mM Tris-HCl, 1 mM EDTA, pH 8.0; Buffer B: 25 mMTris-HCl, 1 mM EDTA, 1 M NaCl, pH 8.0.

Example 239

(Seq. ID No. 218m) C*b¹sAb¹sTb¹sdGsdAsdAsdTsdGsdGsdAsdC*sdC*sAb¹sGb¹sTb¹sAb¹

The compound was synthesized according to the general procedure with theappropriate DNA and LNA building units as exemplified in example 234.After purification, the antisense oligonucleotide was received with apurity of 93.8%. ESI-MS: experimental: 5394.00 Da; calculated: 5393.3Da.

Example 240

(Seq. ID No. 218n) C*b¹Ab¹Tb¹dGsdAsdAsdTsdGsdGsdAsdC*sdC*sAb¹Gb¹Tb¹ Ab¹

The compound was synthesized according to the general procedure with theappropriate DNA building units and LNA building units as exemplified inexample 234 and example 235. After purification, the antisenseoligonucleotide was received with a purity of 94.7%. ESI-MS:experimental: 5297.30 Da; calculated: 5297.0 Da.

Example 241

(Seq. ID No. 218o) C*b¹sAb¹sTb¹sdGsdA*sdA*sdTsdGsdGsdA*sdCsdCsAb¹sGb¹sTb¹sAb¹

The compound was synthesized according to the general procedure with theappropriate DNA and LNA building units as exemplified in example 234.After purification, the antisense oligonucleotide was received with apurity of 92.8%. ESI-MS: experimental: 5410.40 Da; calculated: 5410.3Da.

Example 242

(Seq. ID. No. 218p) C*b¹sAb¹sTb¹sdGsdA*sdA*sdTsdGsdGsdA*sdC*sdC*sAb¹sGb¹sTb¹sAb¹

The compound was synthesized according to the general procedure with theappropriate DNA and LNA building units as exemplified in example 234.After purification, the antisense oligonucleotide was received with apurity of 95.3%. ESI-MS: experimental: 5437.40 Da; calculated: 5438.4Da.

Example 243

(Seq. ID No. 218q) C*b¹sAb¹sTb¹sdGsdAsdAsdTsdGsdGsdAsdC*sdCsAbsGb¹sTb¹sAb¹

The compound was synthesized according to the general procedure with theappropriate DNA and LNA building units as exemplified in example 234.After purification, the antisense oligonucleotide was received with apurity of 93.9%. ESI-MS: experimental: 5378.80 Da; calculated: 5379.3Da.

Example 244

(Seq. ID No. 218c) C*b¹sAb¹sTb¹sdGsdAsdAsdTsdGsdGsdAsdCsdC*sAb¹sGb¹sTb¹sAb¹

The compound was synthesized according to the general procedure with theappropriate DNA and LNA building units as exemplified in example 234.After purification, the antisense oligonucleotide was received with apurity of 92.9%. ESI-MS: experimental: 5379.10 Da; calculated: 5379.3Da.

Example 245

(Seq. ID No. 218s) C*b¹sAb¹sTb¹sdGdAdAdTdGdGdAdC*sdC*sAb¹sGb¹sTb¹ sAb¹

The compound was synthesized according to the general procedure with theappropriate DNA and LNA building units as exemplified in example 234.After purification, the antisense oligonucleotide was received with apurity of 94.5%. ESI-MS: experimental: 5152.70 Da; calculated: 5152.4Da.

Example 246

(Seq. ID No. 218v) /5SpC3/sC*b¹sAb¹sTb¹sdGsdAsdAsdTsdGsdGsdAsdCsdCsAb¹sGb¹sTb¹sAb¹s/3SpC3/

The compound was synthesized according to the general procedure with theappropriate DNA building units and LNA building units as exemplified inexample 234, example 236 and example 237. After purification, theantisense oligonucleotide was received with a purity of 94.4%. ESI-MS:experimental: 5673.50 Da; calculated: 5673.5 Da

Example 247-335

The other oligonucleotides of Table 8 were synthesized according to thegeneral procedure and as shown in the examples. The preparation of theantisense-oligonucleotide including β-D-thio-LNA, α-L-oxy-LNA,β-D-(NH)-LNA, or β-D-(NCH₃)-LNA units were performed in the same way asthe antisense-oligonucleotides containing β-D-oxy-LNA units.

Example 336

(Seq. ID No. 152h) C*b¹sGb¹sAb¹sTb¹sdAsdCsdGsdCsdGsdTsdCsdCsAb¹sC*b¹sAb¹

5′-O-DMT-2′-O,4′-C-methylene-N⁶-benzoyladenosine-3′-O-succinoyl- linkedLCAA CPG (0.2 μmol) was treated with 1400 μl 3% trichloroacetic acid indichloromethane for 60 s to completely remove the 5′-DMT protectiongroup. After several washes with a total amount of 800 μl acetonitrile,the coupling reaction was carried out with 80 μl5′-O-DMT-2′-O—,4′-C-methylene-5-methyl-N⁴-benzoylcytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-O,4′-C-methylene-N⁶-benzoyladenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁴-benzoyl-2′-deoxycytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁴-benzoyl-2′-deoxycytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s. The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-deoxythymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N²-isobutyryl-2′-deoxyguanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁴-benzoyl-2′-deoxycytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N²-isobutyryl-2′-deoxyguanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁴-benzoyl-2′-deoxycytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁶-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl 5′-O-DMT-2′-O,4′-C-methylenethymidine 3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (0.07 M)and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction wasallowed to take place for 250 sec., and excess reagents were flashed outwith 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were insertedto the column for 180 s The system was flushed with 320 μl acetonitrile.For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) were added andallowed to react for 45 sec. At the end of this cycle, the system waswashed with 480 μl acetonitrile. The compound was treated with 1400 μl3% trichloroacetic acid in dichloromethane for 60 s to completely removethe 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-O,4′-C-methylene-N⁶-benzoyladenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-O,4′-C-methylene-N²-dimethyformamidineguanosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-O—,4′-C-methylene-5-methyl-N⁴-benzoylcytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

Upon completion of the solid phase synthesis antisense-oligonucleotideswere treated with a 20% diethylamine solution in acetonitrile (BiosolveBV, Valkenswaard, The Netherlands) for 20 min. to remove the cyanoethylprotecting groups on the phosphate backbone.

Subsequently, the antisense-oligonucleotides were cleaved from the solidsupport and further deprotected using 5 mL concentrated aqueous ammoniafor 16 hours at 55° C. The solid support was separated from theantisense-oligonucleotides by filtration or centrifugation.

Next, the crude antisense-oligonucleotides were purified byanion-exchange high-performance liquid chromatography (HPLC) on an AKTAExplorer System (GE Healthcare, Freiburg, Germany) and a column packedwith Source Q15 (GE Helthcare). Buffer A was 10 mM sodium perchlorate,20 mM Tris, 1 mM EDTA, pH 7.4 and contained 20% acetonitrile and bufferB was the same as buffer A with the exception of 500 mM sodiumperchlorate. A gradient of 15% B to 55% B within 32 column volumes (CV)was employed. UV traces at 280 nm were recorded. Appropriate fractionswere pooled and precipitated with 3 M NaOAc, pH=5.2 and 70% ethanol.Finally, the pellet was washed with 70% ethanol.

Example 337

(Seq. ID No. 152q) C*b ¹ Gb ¹ Ab ¹ Tb ¹dAdCdGdC*dGdTdCdC*Ab ¹ C*b ¹ Ab ¹

The LNA was bound to CPG according to the general procedure. Thecoupling reaction and capping step were also carried out as described inexample 336. After the coupling step, the system was flushed out with800 μl acetonitrile, and 400 μl of 0.02 M Iodine in THF/pyridine/H₂Owere inserted to the column for 45 s. After the oxidation step, thesystem was flushed with 24 μl acetonitrile. After purification, theantisense oligonucleotide was received with a purity of 93.1%.

Example 338

(Seq. ID. No. 152s) /5SpC3s/C*b ¹ sGb ¹ sAb ¹ sTb ¹sdAsdC*sdGsdC*sdGsdTsdCsd CsAb ¹ sC*b ¹ sAb ¹

The compound was synthesized according to the general procedure with theappropriate DNA and LNA building units as exemplified in example 336.But with the exception that after the last nucleotide has been coupledto the oligonucleotide and the subsequent oxidation and capping stepswere carried out, 80 μl of phosphoramidite-C₃ (0.07 M) and 236 μl DCI inacetonitrile (0.25 M) were added.

The coupling was allowed to take place for 250 sec., and excess reagentswere flashed out with 800 μl acetonitrile. The subsequent steps wereperformed as described in example 336. After purification, the antisenseoligonucleotide was received with a purity of 96.5%.

Example 339

(Seq. ID No. 152t) C*b ¹ sGb ¹ sAb ¹ sTb ¹ sdAsdC*sdGsdCsdGsdTsdCsdC*sAb¹ sC* b ¹ sAb ¹/3SpC3s/

3′-Spacer C3 CPG (0.2 μmol) was treated with 1400 μl 3% trichloroaceticacid in dichloromethane for 60 s to completely remove the 5′-DMTprotection group. After several washes with a total amount of 800 μlacetonitrile, the coupling reaction was carried out with 80 μl5′-O-DMT-2′-O,4′-C-methylene-N⁶-benzoyladenosine-3′-[(2-cyanoethyl-N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group. The subsequent reactionswere performed as described in example 336. After purification, theantisense oligonucleotide was received with a purity of 92.1%.

Example 340

(Seq. ID No. 152aa) C*b ¹ ssGb ¹ssAb¹ssdTssdAssdC*ssdGssdCssdGssdTssdCssd C*ssAb ¹ ssC*b ¹ ssAb ¹

5′-O-DMT-2′-O,4′-C-methylene-N⁶-benzoyladenosine-3′-O-succinoyl- linkedLCAA CPG (0.2 μmol) was treated with 1400 μl 3% trichloroacetic acid indichloromethane for 60 s to completely remove the 5′-DMT protectiongroup. After several washes with a total amount of 800 μl acetonitrile,the coupling reaction was carried out with 38 μl5′-O-DMT-2′-O—,4′-C-methylene-5-methyl-N⁴-benzoylcytidine-3′-[(β-benzoylmercapto)ethyl]pyrrolidinolthiophosphoramidite(0.15 M) in 10% dichloromethane (v/V) in acetonitrile and 236 μl DCI inacetonitrile (0.25 M). The coupling reaction was allowed to take placefor 250 sec., and excess reagents were flashed out with 800 μlacetonitrile, and 900 μl of DDTT (0.05 M in pyridine/acetonitrile 4:1v/v) were inserted to the column for 240 s The system was flushed with320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride inTHF (1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1)were added and allowed to react for 45 sec. At the end of this cycle,the system was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 38 μl5′-O-DMT-2′-O,4′-C-methylene-N⁶-benzoyladenosine-3′-[(3-benzoylmercapto)ethyl]pyrrolidinolthiophosphoramidite(0.15 M) in 10% dichloromethane (v/v) in acetonitrile and 236 μl DCI inacetonitrile (0.25 M). The coupling reaction was allowed to take placefor 250 sec., and excess reagents were flashed out with 800 μlacetonitrile, and 900 μl of DDTT (0.05 M in pyridine/acetonitrile 4:1v/v) were inserted to the column for 240 s The system was flushed with320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride inTHF (1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1)were added and allowed to react for 45 sec. At the end of this cycle,the system was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The further elongation of the oligonucleotide was performed in the sameway.

Upon completion of the solid phase synthesis, theantisense-oligonucleotides were treated 850 μl ammonia in concentratedethanol (ammonia/ethanol 3:1 v/v) at 55° C. for 15-16h in order tocleave antisense-oligonucleotide from the solid-support and to deprotectthe thiol-group.

Next, the crude antisense-oligonucleotide was purified by anion-exchangechromatogtraophy using a Mono Q 10/100 GL column. The buffers wereprepared with DEPC-treated water, and their compositions were asfollows: Buffer A: 25 mM Tris-HCl, 1 mM EDTA, pH 8.0; Buffer B: 25 mMTris-HCl, 1 mM EDTA, 1 M NaCl, pH 8.0.

Example 341-433

The other oligonucleotides of Table 5 were synthesized according to thegeneral procedure and as shown in the examples. The preparation of theantisense-oligonucleotide including β-D-thio-LNA, α-L-oxy-LNA,β-D-(NH)-LNA, or β-D-(NCH₃)-LNA units were performed in the same way asthe antisense-oligonucleotides containing β-D-oxy-LNA units.

Example 434

(Seq. ID No. 143h) C*b¹sTb¹sdCsdGsdTsdCsdAsdTsdAsdGsdAsC*b¹sC*b¹sGb¹

5′-O-DMT-2′-O,4′-C-methylene-N²-dimethyformamidineguanosine-3′-O-succinoyl-linked LCAA CPG (0.2 μmol) was treated with 1400 μl 3%trichloroacetic acid in dichloromethane for 60 s to completely removethe 5′-DMT protection group. After several washes with a total amount of800 μl acetonitrile, the coupling reaction was carried out with 80 μl5′-O-DMT-2′-O—,4′-C-methylene-5-methyl-N⁴-benzoylcytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-O—,4′-C-methylene-5-methyl-N⁴-benzoylcytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁶-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N²-isobutyryl-2′-deoxyguanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁶-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-deoxythymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁶-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁴-benzoyl-2′-deoxycytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-deoxythymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N²-isobutyryl-2′-deoxyguanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁴-benzoyl-2′-deoxycytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl 5′-O-DMT-2′-O,4′-C-methylenethymidine 3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (0.07 M)and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction wasallowed to take place for 250 sec., and excess reagents were flashed outwith 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were insertedto the column for 180 s The system was flushed with 320 μl acetonitrile.For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) were added andallowed to react for 45 sec. At the end of this cycle, the system waswashed with 480 μl acetonitrile. The compound was treated with 1400 μl3% trichloroacetic acid in dichloromethane for 60 s to completely removethe 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-O—,4′-C-methylene-5-methyl-N⁴-benzoylcytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

Upon completion of the solid phase synthesis antisense-oligonucleotideswere treated with a 20% diethylamine solution in acetonitrile (BiosolveBV, Valkenswaard, The Netherlands) for 20 min. to remove the cyanoethylprotecting groups on the phosphate backbone.

Subsequently, the antisense-oligonucleotides were cleaved from the solidsupport and further deprotected using 5 mL concentrated aqueous ammoniafor 16 hours at 55° C. The solid support was separated from theantisense-oligonucleotides by filtration or centrifugation.

Next, the crude antisense-oligonucleotides were purified byanion-exchange high-performance liquid chromatography (HPLC) on an AKTAExplorer System (GE Healthcare, Freiburg, Germany) and a column packedwith Source Q15 (GE Helthcare). Buffer A was 10 mM sodium perchlorate,20 mM Tris, 1 mM EDTA, pH 7.4 and contained 20% acetonitrile and bufferB was the same as buffer A with the exception of 500 mM sodiumperchlorate. A gradient of 15% B to 55% B within 32 column volumes (CV)was employed. UV traces at 280 nm were recorded. Appropriate fractionswere pooled and precipitated with 3 M NaOAc, pH=5.2 and 70% ethanol.Finally, the pellet was washed with 70% ethanol.

Example 435

(Seq. ID No. 143ad) C*b ¹ Tb ¹dC*dGdTdCdAdTdAdGdAC*b ¹ C*b ¹ Gb ¹

The LNA was bound to CPG according to the general procedure. Thecoupling reaction and capping step were also carried out as described inexample 434. After the capping step, the system was flushed out with 800μl acetonitrile, and 400 μl of 0.02 M Iodine in THF/pyridine/H₂O wereinserted to the column for 45 s. The system was flushed after theoxidation step with 24 μl acetonitrile. After purification, theantisense oligonucleotide was received with a purity of 88.7%.

Example 436

(Seq. ID No. 143af) /5SpC3s/C*b ¹ sTb ¹ sdC*dGdTdC*dA*dTdAdGdA*sC*b ¹sC*b ¹ s Gb ¹

The compound was synthesized according to the general procedure with theappropriate DNA and LNA building units as exemplified in example 434 andexample 435. But with the exception that after the last nucleotide hasbeen coupled to the oligonucleotide and the subsequent oxidation andcapping steps were carried out, 80 μl of phosphoramidite-C3 (0.07 M) and236 μl DCI in acetonitrile (0.25 M) were added. The coupling was allowedto take place for 250 sec., and excess reagents were flashed out with800 μl acetonitrile. The subsequent steps were performed as described inexample 434 and example 435. After purification, the antisenseoligonucleotide was received with a purity of 94.4%.

Example 437

(Seq. ID No. 143ag) C*b ¹ sTb ¹ sdC*dGdTdC*dA*dTdAdGdA*sC*b ¹ sC*b ¹ sGb¹/ 3SpC3s/

3′-Spacer C3 CPG (0.2 μmol) was treated with 1400 μl 3% trichloroaceticacid in dichloromethane for 60 s to completely remove the 5′-DMTprotection group. After several washes with a total amount of 800 μlacetonitrile, the coupling reaction was carried out with 80 μl5′-O-DMT-2′-O,4′-C-methylene-N²-diemthyformamidineguanosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group. The subsequent reactionswere performed as described in example 434 and example 435. Afterpurification, the antisense oligonucleotide was received with a purityof 91.6%.

Example 438

(Seq. ID No. 143t) C*b ¹ ssTb ¹ ssC*b ¹ssdGssdTssdC*ssdAssdTssdAssdGssdAss C*b ¹ ssC*b ¹ ssGb ¹

5′-O-DMT-2′-O,4′-C-methylene-N²-dimethyformamidineguanosine-3′-O-succinoyl-linkedLCAA CPG (0.2 μmol) was treated with 1400 μl 3% trichloroacetic acid indichloromethane for 60 s to completely remove the 5′-DMT protectiongroup. After several washes with a total amount of 800 μl acetonitrile,the coupling reaction was carried out with 38 μl5′-O-DMT-2′-O—,4′-C-methylene-5-methyl-N⁴-benzoylcytidine-3′-[(β-benzoylmercapto)ethyl]pyrrolidinolthiophosphoramidite(0.15 M) in 10% dichloromethane (v/V) in acetonitrile and 236 μl DCI inacetonitrile (0.25 M). The coupling reaction was allowed to take placefor 250 sec., and excess reagents were flashed out with 800 μlacetonitrile, and 900 μl of DDTT (0.05 M in pyridine/acetonitrile 4:1v/v) were inserted to the column for 240 s The system was flushed with320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride inTHF (1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1)were added and allowed to react for 45 sec. At the end of this cycle,the system was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 38 μl5′-O-DMT-2′-O—,4′-C-methylene-5-methyl-N⁴-benzoylcytidine-3′-[(β-benzoylmercapto)ethyl]pyrrolidinolthiophosphoramidite(0.15 M) in 10% dichloromethane (v/v) in acetonitrile and 236 μl DCI inacetonitrile (0.25 M). The coupling reaction was allowed to take placefor 250 sec., and excess reagents were flashed out with 800 μlacetonitrile, and 900 μl of DDTT (0.05 M in pyridine/acetonitrile 4:1v/v) were inserted to the column for 240 s The system was flushed with320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride inTHF (1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1)were added and allowed to react for 45 sec. At the end of this cycle,the system was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The further elongation of the oligonucleotide was performed in the sameway.

Upon completion of the solid phase synthesis, theantisense-oligonucleotides were treated 850 μl ammonia in concentratedethanol (ammonia/ethanol 3:1 v/v) at 55° C. for 15-16h in order tocleave antisense-oligonucleotide from the solid-support and to deprotectthe thiol-group.

Next, the crude antisense-oligonucleotide was purified by anion-exchangechromatogtraophy using a Mono Q 10/100 GL column. The buffers wereprepared with DEPC-treated water, and their compositions were asfollows: Buffer A: 25 mM Tris-HCl, 1 mM EDTA, pH 8.0; Buffer B: 25 mMTris-HCl, 1 mM EDTA, 1 M NaCl, pH 8.0.

Example 439-534

(Seq. ID No. 213k) C*b¹sAb¹sGb¹sdGsdCsdAsdTsdTsdAsdAsdTsdAsdAsdAsGb¹sTb¹sGb¹

The other oligonucleotides of Table 4 were synthesized according to thegeneral procedure and as shown in the examples. The preparation of theantisense-oligonucleotide including β-D-thio-LNA, α-L-oxy-LNA,β-D-(NH)-LNA, or β-D-(NCH₃)-LNA units were performed in the same way asthe antisense-oligonucleotides containing β-D-oxy-LNA units.

Example 535

5′-O-DMT-2′-O,4′-C-methylene-N²-dimethyformamidineguanosine-3′-O-succinoyl-linkedLCAA CPG (0.2 μmol) was treated with 1400 μl 3% trichloroacetic acid indichloromethane for 60 s to completely remove the 5′-DMT protectiongroup. After several washes with a total amount of 800 μl acetonitrile,the coupling reaction was carried out with 80 μl5′-O-DMT-2′-O,4′-C-methylene thymidine3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (0.07 M) and 236μl DCI in acetonitrile (0.25 M).

The coupling reaction was allowed to take place for 250 sec., and excessreagents were flashed out with 800 μl acetonitrile, and 640 μl ofBeaucage (0.2 M) were inserted to the column for 180 s The system wasflushed with 320 μl acetonitrile. For the capping step, 448 μl of aceticanhydride in THF (1:9 v/v) and 448 μl N-methylimidazol(NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec.At the end of this cycle, the system was washed with 480 μlacetonitrile. The compound was treated with 1400 μl 3% trichloroaceticacid in dichloromethane for 60 s to completely remove the 5′-DMTprotection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-O,4′-C-methylene-N²-dimethyformamidineguanosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁶-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁶-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁶-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-deoxythymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁶-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁶-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-deoxythymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-deoxythymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁶-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N⁴-benzoyl-2′-deoxycytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-N²-isobutyryl-2′-deoxyguanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-O,4′-C-methylene-N²-dimethyformamidineguanosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-O,4′-C-methylene-N⁶-benzoyladenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s. The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 80 μl5′-O-DMT-2′-O—,4′-C-methylene-5-methyl-N⁴-benzoylcytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

Upon completion of the solid phase synthesis antisense-oligonucleotideswere treated with a 20% diethylamine solution in acetonitrile (BiosolveBV, Valkenswaard, The Netherlands) for 20 min. to remove the cyanoethylprotecting groups on the phosphate backbone.

Subsequently, the antisense-oligonucleotides were cleaved from the solidsupport and further deprotected using 5 mL concentrated aqueous ammoniafor 16 hours at 55° C. The solid support was separated from theantisense-oligonucleotides by filtration or centrifugation.

Next, the crude antisense-oligonucleotides were purified byanion-exchange high-performance liquid chromatography (HPLC) on an AKTAExplorer System (GE Healthcare, Freiburg, Germany) and a column packedwith Source Q15 (GE Helthcare). Buffer A was 10 mM sodium perchlorate,20 mM Tris, 1 mM EDTA, pH 7.4 and contained 20% acetonitrile and bufferB was the same as buffer A with the exception of 500 mM sodiumperchlorate. A gradient of 15% B to 55% B within 32 column volumes (CV)was employed. UV traces at 280 nm were recorded. Appropriate fractionswere pooled and precipitated with 3 M NaOAc, pH=5.2 and 70% ethanol.Finally, the pellet was washed with 70% ethanol.

Example 536

(Seq. ID No. 213n) C*b ¹ Ab ¹ Gb ¹dGdC*dAdTdTdAdAdTdAdAdAGb ¹ Tb ¹ Gb ¹

The LNA was bound to CPG according to general procedure. The couplingreaction and capping step were also carried out as described in example535. After the capping step, the system was flushed out with 800 μlacetonitrile, and 400 μl of 0.02 M Iodine in THF/pyridine/H₂O wereinserted to the column for 45 s. The system was flushed after theoxidation step with 24 μl acetonitrile. After purification, theantisense oligonucleotide was received with a purity of 91.4%.

Example 537

(Seq. ID No. 213o) /5SpC3s/C*b ¹ ¹ sGb ¹ sdGsdC*sdAsdTsdTsdAsdAsdTsdAsdAsdAsGb ¹ sTb ¹ sGb ¹

The compound was synthesized according to the general procedure with theappropriate DNA and LNA building units as exemplified in example 535.But with the exception that after the last nucleotide has been coupledto the oligonucleotide and the subsequent oxidation and capping stepswere carried out, 80 μl of phosphoramidite-C₃ (0.07 M) and 236 μl DCI inacetonitrile (0.25 M) were added. The coupling was allowed to take placefor 250 sec., and excess reagents were flashed out with 800 μlacetonitrile. The subsequent steps were performed as described inexample 535. After purification, the antisense oligonucleotide wasreceived with a purity of 87.1%.

Example 538

(Seq. ID No. 213p) C*b ¹ sAb ¹ sGb ¹sdGsdC*sdAsdTsdTsdAsdAsdTsdAsdAsdAsGb ¹ sTb ¹ sGb ¹/3SpC3s/

3′-Spacer C3 CPG (0.2 μmol) was treated with 1400 μl 3% trichloroaceticacid in dichloromethane for 60 s to completely remove the 5′-DMTprotection group. After several washes with a total amount of 800 μlacetonitrile, the coupling reaction was carried out with 80 μl5′-O-DMT-2′-O,4′-C-methylene-N²-diemthyformamidineguanosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reactionwas allowed to take place for 250 sec., and excess reagents were flashedout with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) wereinserted to the column for 180 s The system was flushed with 320 μlacetonitrile. For the capping step, 448 μl of acetic anhydride in THF(1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) wereadded and allowed to react for 45 sec. At the end of this cycle, thesystem was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group. The subsequent reactionswere performed as described in example 535. After purification, theantisense oligonucleotide was received with a purity of 95.7%.

Example 539

(Seq. ID No. 213ae) C*b ¹ ssAb ¹ ssGb ¹ssdGssdC*ssdAssdTssdTssdAssdAssdTssd AssdAssAb ¹ ssGb ¹ ssTb ¹ ssGb ¹

5′-O-DMT-2′-O,4′-C-methylene-N²-dimethyformamidineguanosine-3′-O-succinoyl-linkedLCAA CPG (0.2 μmol) was treated with 1400 μl 3% trichloroacetic acid indichloromethane for 60 s to completely remove the 5′-DMT protectiongroup. After several washes with a total amount of 800 μl acetonitrile,the coupling reaction was carried out with 38 μl5′-O-DMT-2′-O,4′-C-methylene-thymidine-3′-[(β-benzoylmercapto)ethyl]pyrrolidinolthiophosphoramidite(0.15 M) in 10% dichloromethane (v/V) in acetonitrile and 236 μl DCI inacetonitrile (0.25 M). The coupling reaction was allowed to take placefor 250 sec., and excess reagents were flashed out with 800 μlacetonitrile, and 900 μl of DDTT (0.05 M in pyridine/acetonitrile 4:1v/v) were inserted to the column for 240 s The system was flushed with320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride inTHF (1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1)were added and allowed to react for 45 sec. At the end of this cycle,the system was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The coupling was carried out with 38 μl5′-O-DMT-2′-O,4′-C-methylene-N²-dimethyformamidineguanosine-3′-[(β-benzoylmercapto)ethyl]pyrrolidinolthiophosphoramidite(0.15 M) in 10% dichloromethane (v/v) in acetonitrile and 236 μl DCI inacetonitrile (0.25 M). The coupling reaction was allowed to take placefor 250 sec., and excess reagents were flashed out with 800 μlacetonitrile, and 900 μl of DDTT (0.05 M in pyridine/acetonitrile 4:1v/v) were inserted to the column for 240 s The system was flushed with320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride inTHF (1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1)were added and allowed to react for 45 sec. At the end of this cycle,the system was washed with 480 μl acetonitrile. The compound was treatedwith 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s tocompletely remove the 5′-DMT protection group.

The further elongation of the oligonucleotide was performed in the sameway.

Upon completion of the solid phase synthesis, theantisense-oligonucleotides were treated 850 μl ammonia in concentratedethanol (ammonia/ethanol 3:1 v/v) at 55° C. for 15-16h in order tocleave antisense-oligonucleotide from the solid-support and to deprotectthe thiol-group.

Next, the crude antisense-oligonucleotide was purified by anion-exchangechromatogtraophy using a Mono Q 10/100 GL column. The buffers wereprepared with DEPC-treated water, and their compositions were asfollows: Buffer A: 25 mM Tris-HCl, 1 mM EDTA, pH 8.0; Buffer B: 25 mMTris-HCl, 1 mM EDTA, 1 M NaCl, pH 8.0.

Examples 540-640

The other oligonucleotides of Table 9 were synthesized according to thegeneral procedure and as shown in the examples. The preparation of theantisense-oligonucleotide including β-D-thio-LNA, α-L-oxy-LNA,β-D-(NH)-LNA, or β-D-(NCH₃)-LNA units were performed in the same way asthe antisense-oligonucleotides containing β-D-oxy-LNA units.

Sequence Listing Seq. ID No. 1: Homo sapiens transforming growth factor,beta receptor II (TGFBR2), transcript variant 2, (antisense; DNA code)TTTAGCTACT AGGAATGGGA ACAGGAGGCA GGATGCTCAC CTGAGTATTT TGCTTTATTC 60AATCTAATAA ACATTTTATT TATGTAAAAG ACAAACAATG CATAGAATAA AAATAAGTGC 120TTGAGACTTT TGATATAAAA AGAGTATATA GCATTCACAT TCCTATTTTA ATACATGAGT 180ACAGCTGAAG TGTTCCATAA AAGAATAAAA CTTTCCCTTT ATGTATAGTA GTGAAAAAAG 240TCAGTATTTT TAGGAACTAC AGAATGTTAT TCCTTGGTCT TTTTTCTTGA ATAAGAAAAA 300AAAACATAAA CAAAACAAGC CACAGTATCC TCTGACACTA CATTCCAGTT TATGCTGATA 360ACCCAGAAGT GAGAATACTC TTGAATCTTG AATATCTCAT GAATGGACCA GTATTCTAGA 420AACTCACCAC TAGAGGTCAA TGGGCAACAG CTATTGGGAT GGTATCAGCA TGCCCTACGG 480TGCAAGTGGA ATTTCTAGGC GCCTCTATGC TACTGCAGCC ACACTGTCTT TAACTCTCAG 540CCCACCCACA CTGAGGAGGG TGCCTAGAGG TTCTATTTCC AAACCTTTGC ATGTATCTTA 600AAAATCTCAA TAAAATGAGA CCTTCCACCA TCCAAACAGA GCTGATATTC TCACTACCAG 660TCCCTCTCTA ATATTCCTAT TTGGCTGAAA ATAAGTAGCT TCAAAAAGTT TTAAAAAAGA 720GATTACTTGC AGCATTAACA CTTCTTTGTT GATTAACAAG TTTCCTATGG AGTTTTAAAG 780CTCATACTTT GTTCTTGTCC TTGTGGACAC AAATTTTCTA ACTGCAAATG GGACCTTTGT 840GTCCCACATT CAAATCCTCT CTAGTAATTT CTGCAAAGGT TGAGAAGGCT GGCATGATGG 900AGAGAACGGT AACCATGAGG AAAGCTTCTT GGAGTAAAGC ACTCCTCTCT CCAATGCAGA 960GGGTAAAACT ATTAACATAT AAGCAAAAGA AACTTGGGCT AACTGAGACC CTTAAAGGAG 1020TTCCCCTTTA GTCCAATAAA AGGCCAACTT CAAATCTTAA CACCAGATAA GGTAGTCAAA 1080ATCATATTAT ATACCCAGAG AATGACTGCT TGAATGGACA TTTCTTACAA GGGACCTTGG 1140TTAGGTGCAG ATTTAATTCC TAGACTGGGG TCCAGGTAGG CAGTGGAAAG AGCTAATGTT 1200TACAGTGAGA AGTGAGGCAG CTTTGTAAGT GTCTCCACAC CTTCACATTT TGTGAACGTG 1260GACTGGAGAT AACTGAAAAC CATCTGCTAT CCTTACCTGG GGATCCAGAT TTTCCTGCAA 1320AATCTCCAAA TATTTATAAA GTGGCTTCAC TTTTTGAAAC GCTGTGCTGA CCAAACAAAA 1380CATATGTTTA GAGTGCCTGA GGTCATAGTC CTGACAATGA TAGTATTGTG TAGTTGAAAT 1440CCTCTTCATC AGGCCAAACT GTGCTTGAGC AATCAGGAGC CCAGAAAGAT GGAACCCATT 1500GGTGTTTGTA TAGAAAACTA GAAAATCAAG TCAAGTGTAA TGAAAAAGTA AACACGATAA 1560AGCCTAGAGT GAGAATTTGC TCCTTTTTAG AAAAGGATGA AGGCTGGGAG CAGAGAATAG 1620TAACATAAGT GCAGGGGAAA GATGAAAAAA AGAACAATTT TTCATTAGTA GATGGTGGGG 1680CAATCGCATG GATGGGGACA TCTGTTCTGA TTTTTCTGCA ACCCATGAAG GTAAAAAGTG 1740GGGTTCAAAA CATTCAAGGT ATTAAAGATG GGGTAGAGTT TCTAAACTAG GTTGAGGGAG 1800AGTTTCTAAA CTAGCCCCCC AGATTTGGGG CTTGGAGCTT AAATGAAAAG TCCAGGAGAA 1860ATAAGGGCAC ACAGGAACCC CGGGAACACT GGTCCTCAAA CAGTGCCACT GTACTTAGTT 1920CCATGGCCAG AAGAGAAGTG CTAGGCAGGG AATGATTATT TTGCAAAAGC AAGTGCAATG 1980TGGTCATAGC TGGCTGTGAG ACATGGAGCC TCTTTCCTCA TGCAAAGTTC ACTGTTTTAC 2040AGTCAGAGAA CCACTGCATG TGTGATTGTC AAATGCTAAT GCTGTCATGG GTCCCTTCCT 2100TCTCTGCTTG GTTCTGGAGT TCTCCAATAA AACCAATTTC CTGGGAATAT TTGATGTTTT 2160TCCTTGTCTC TTTTCAAGGT ATGGCTATAT ATATAGAGCT ATAGACATAT ATAGATATAT 2220ATATATATAT ATAAAACATA GCTATTCATA TTTATATACA GGCATTAATA AAGTGCAAAT 2280GTTATTGGCT ATTGTAAAAA TCAATCTCAT TTCCTGAGGA AGTGCTAACA CAGCTTATCC 2340TATGACAATG TCAAAGGCAT AGAATGCTCT ATGTCACCCA CTCCCTGCTG CTGTTGTTTC 2400TGCTTATCCC CACAGCTTAC AGGGAGGGGA GTGACCCCCT TGGTTTTCCA GGAAGCATCA 2460GTTCAGGGGC AGCTTCCTGC TGCCTCTGTT CTTTGGTGAG AGGGGCAGCC TCTTTGGACA 2520TGGCCCAGCC TGCCCCAGAA GAGCTATTTG GTAGTGTTTA GGGAGCCGTC TTCAGGAATC 2580TTCTCCTCCG AGCAGCTCCT CCCCGAGAGC CTGTCCAGAT GCTCCAGCTC ACTGAAGCGT 2640TCTGCCACAC ACTGGGCTGT GAGACGGGCC TCTGGGTCGT GGTCCCAGCA CTCAGTCAAC 2700GTCTCACACA CCATCTGGAT GCCCTGGTGG TTGAGCCAGA AGCTGGGAAT TTCTGGTCGC 2760CCTCGATCTC TCAACACGTT GTCCTTCATG CTTTCGACAC AGGGGTGCTC CCGCACCTTG 2820GAACCAAATG GAGGCTCATA ATCTTTTACT TCTCCCACTG CATTACAGCG AGATGTCATT 2880TCCCAGAGCA CCAGAGCCAT GGAGTAGACA TCGGTCTGCT TGAAGGACTC AACATTCTCC 2940AAATTCATCC TGGATTCTAG GACTTCTGGA GCCATGTATC TTGCAGTTCC CACCTGCCCA 3000CTGTTAGCCA GGTCATCCAC AGACAGAGTA GGGTCCAGAC GCAGGGAAAG CCCAAAGTCA 3060CACAGGCAGC AGGTTAGGTC GTTCTTCACG AGGATATTGG AGCTCTTGAG GTCCCTGTGC 3120ACGATGGGCA TCTTGGGCCT CCCACATGGA GTGTGATCAC TGTGGAGGTG AGCAATCCCC 3180CGGGCGAGGG AGCTGCCCAG CTTGCGCAGG TCCTCCCAGC TGATGACATG CCGCGTCAGG 3240TACTCCTGTA GGTTGCCCTT GGCGTGGAAG GCGGTGATCA GCCAGTATTG TTTCCCCAAC 3300TCCGTCTTCC GCTCCTCAGC CGTCAGGAAC TGGAGTATGT TCTCATGCTT CAGATTGATG 3360TCTGAGAAGA TGTCCTTCTC TGTCTTCCAA GAGGCATACT CCTCATAGGG AAAGATCTTG 3420ACTGCCACTG TCTCAAACTG CTCTGAAGTG TTCTGCTTCA GCTTGGCCTT ATAGACCTCA 3480GCAAAGCGAC CTTTCCCCAC CAGGGTGTCC AGCTCAATGG GCAGCAGCTC TGTGTTGTGG 3540TTGATGTTGT TGGCACACGT GGAGCTGATG TCAGAGCGGT CATCTTCCAG GATGATGGCA 3600CAGTGCTCGC TGAACTCCAT GAGCTTCCGC GTCTTGCCGG TTTCCCAGGT TGAACTCAGC 3660TTCTGCTGCC GGTTAACGCG GTAGCAGTAG AAGATGATGA TGACAGATAT GGCAACTCCC 3720AGTGGTGGCA GGAGGCTGAT GCCTGTCACT TGAAATATGA CTAGCAACAA GTCAGGATTG 3780CTGGTGTTAT ATTCTTCTGA GAAGATGATG TTGTCATTGC ACTCATCAGA GCTACAGGAA 3840CACATGAAGA AAGTCTCACC AGGCTTTTTT TTTTCCTTCA TAATGCACTT TGGAGAAGCA 3900GCATCTTCCA GAATAAAGTC ATGGTAGGGG AGCTTGGGGT CATGGCAAAC TGTCTCTAGT 3960GTTATGTTCT CGTCATTCTT TCTCCATACA GCCACACAGA CTTCCTGTGG CTTCTCACAG 4020ATGGAGGTGA TGCTGCAGTT GCTCATGCAG GATTTCTGGT TGTCACAGGT GGAAAATCTC 4080ACATCACAAA ATTTACACAG TTGTGGAAAC TTGACTGCAC CGTTGTTGTC AGTGACTATC 4140ATGTCGTTAT TAACCGACTT CTGAACGTGC GGTGGGATCG TGCTGGCGAT ACGCGTCCAC 4200AGGACGATGT GCAGCGGCCA CAGGCCCCTG AGCAGCCCCC GACCCATGGC AGACCCCGCT 4260GCTCGTCATA GACCGAGCCC CCAGCGCAGC GGACGGCGCC TTCCCGGACC CCTGGCTGCG 4320CCTCCGCGCC GCGCCCTCTC CGGACCCCGC GCCGGGCCGG CAGCGCAGAT GTGCGGGCCA 4380GATGTGGCGC CCGCTCGCCA GCCAGGAGGG GGCCTGGAGG CCGGCGAGGC GCGGGGAGGC 4440CCCCGGCGGC CGAGGGAAGC TGCACAGGAG TCCGGCTCCT GTCCCGAGCG GGTGCACGCG 4500CGGGGGTGTC GTCGCTCCGT GCGCGCGAGT GACTCACTCA ACTTCAACTC AGCGCTGCGG 4560GGGAAACAGG AAACTCCTCG CCAACAGCTG GGCAGGACCT CTCTCCGCCC GAGAGCCTTC 4620TCCCTCTCC 4629 Seq. ID No. 2: Homo sapiens transforming growth factor,beta receptor II (TGFBR2), transcript variant 2, mRNA (sense; written inDNA code) GGAGAGGGAG AAGGCTCTCG GGCGGAGAGA GGTCCTGCCC AGCTGTTGGCGAGGAGTTTC 60 CTGTTTCCCC CGCAGCGCTG AGTTGAAGTT GAGTGAGTCA CTCGCGCGCACGGAGCGACG 120 ACACCCCCGC GCGTGCACCC GCTCGGGACA GGAGCCGGAC TCCTGTGCAGCTTCCCTCGG 180 CCGCCGGGGG CCTCCCCGCG CCTCGCCGGC CTCCAGGCCC CCTCCTGGCTGGCGAGCGGG 240 CGCCACATCT GGCCCGCACA TCTGCGCTGC CGGCCCGGCG CGGGGTCCGGAGAGGGCGCG 300 GCGCGGAGGC GCAGCCAGGG GTCCGGGAAG GCGCCGTCCG CTGCGCTGGGGGCTCGGTCT 360 ATGACGAGCA GCGGGGTCTG CCATGGGTCG GGGGCTGCTC AGGGGCCTGTGGCCGCTGCA 420 CATCGTCCTG TGGACGCGTA TCGCCAGCAC GATCCCACCG CACGTTCAGAAGTCGGTTAA 480 TAACGACATG ATAGTCACTG ACAACAACGG TGCAGTCAAG TTTCCACAACTGTGTAAATT 540 TTGTGATGTG AGATTTTCCA CCTGTGACAA CCAGAAATCC TGCATGAGCAACTGCAGCAT 600 CACCTCCATC TGTGAGAAGC CACAGGAAGT CTGTGTGGCT GTATGGAGAAAGAATGACGA 660 GAACATAACA CTAGAGACAG TTTGCCATGA CCCCAAGCTC CCCTACCATGACTTTATTCT 720 GGAAGATGCT GCTTCTCCAA AGTGCATTAT GAAGGAAAAA AAAAAGCCTGGTGAGACTTT 780 CTTCATGTGT TCCTGTAGCT CTGATGAGTG CAATGACAAC ATCATCTTCTCAGAAGAATA 840 TAACACCAGC AATCCTGACT TGTTGCTAGT CATATTTCAA GTGACAGGCATCAGCCTCCT 900 GCCACCACTG GGAGTTGCCA TATCTGTCAT CATCATCTTC TACTGCTACCGCGTTAACCG 960 GCAGCAGAAG CTGAGTTCAA CCTGGGAAAC CGGCAAGACG CGGAAGCTCATGGAGTTCAG 1020 CGAGCACTGT GCCATCATCC TGGAAGATGA CCGCTCTGAC ATCAGCTCCACGTGTGCCAA 1080 CAACATCAAC CACAACACAG AGCTGCTGCC CATTGAGCTG GACACCCTGGTGGGGAAAGG 1140 TCGCTTTGCT GAGGTCTATA AGGCCAAGCT GAAGCAGAAC ACTTCAGAGCAGTTTGAGAC 1200 AGTGGCAGTC AAGATCTTTC CCTATGAGGA GTATGCCTCT TGGAAGACAGAGAAGGACAT 1260 CTTCTCAGAC ATCAATCTGA AGCATGAGAA CATACTCCAG TTCCTGACGGCTGAGGAGCG 1320 GAAGACGGAG TTGGGGAAAC AATACTGGCT GATCACCGCC TTCCACGCCAAGGGCAACCT 1380 ACAGGAGTAC CTGACGCGGC ATGTCATCAG CTGGGAGGAC CTGCGCAAGCTGGGCAGCTC 1440 CCTCGCCCGG GGGATTGCTC ACCTCCACAG TGATCACACT CCATGTGGGAGGCCCAAGAT 1500 GCCCATCGTG CACAGGGACC TCAAGAGCTC CAATATCCTC GTGAAGAACGACCTAACCTG 1560 CTGCCTGTGT GACTTTGGGC TTTCCCTGCG TCTGGACCCT ACTCTGTCTGTGGATGACCT 1620 GGCTAACAGT GGGCAGGTGG GAACTGCAAG ATACATGGCT CCAGAAGTCCTAGAATCCAG 1680 GATGAATTTG GAGAATGTTG AGTCCTTCAA GCAGACCGAT GTCTACTCCATGGCTCTGGT 1740 GCTCTGGGAA ATGACATCTC GCTGTAATGC AGTGGGAGAA GTAAAAGATTATGAGCCTCC 1800 ATTTGGTTCC AAGGTGCGGG AGCACCCCTG TGTCGAAAGC ATGAAGGACAACGTGTTGAG 1860 AGATCGAGGG CGACCAGAAA TTCCCAGCTT CTGGCTCAAC CACCAGGGCATCCAGATGGT 1920 GTGTGAGACG TTGACTGAGT GCTGGGACCA CGACCCAGAG GCCCGTCTCACAGCCCAGTG 1980 TGTGGCAGAA CGCTTCAGTG AGCTGGAGCA TCTGGACAGG CTCTCGGGGAGGAGCTGCTC 2040 GGAGGAGAAG ATTCCTGAAG ACGGCTCCCT AAACACTACC AAATAGCTCTTCTGGGGCAG 2100 GCTGGGCCAT GTCCAAAGAG GCTGCCCCTC TCACCAAAGA ACAGAGGCAGCAGGAAGCTG 2160 CCCCTGAACT GATGCTTCCT GGAAAACCAA GGGGGTCACT CCCCTCCCTGTAAGCTGTGG 2220 GGATAAGCAG AAACAACAGC AGCAGGGAGT GGGTGACATA GAGCATTCTATGCCTTTGAC 2280 ATTGTCATAG GATAAGCTGT GTTAGCACTT CCTCAGGAAA TGAGATTGATTTTTACAATA 2340 GCCAATAACA TTTGCACTTT ATTAATGCCT GTATATAAAT ATGAATAGCTATGTTTTATA 2400 TATATATATA TATATCTATA TATGTCTATA GCTCTATATA TATAGCCATACCTTGAAAAG 2460 AGACAAGGAA AAACATCAAA TATTCCCAGG AAATTGGTTT TATTGGAGAACTCCAGAACC 2520 AAGCAGAGAA GGAAGGGACC CATGACAGCA TTAGCATTTG ACAATCACACATGCAGTGGT 2580 TCTCTGACTG TAAAACAGTG AACTTTGCAT GAGGAAAGAG GCTCCATGTCTCACAGCCAG 2640 CTATGACCAC ATTGCACTTG CTTTTGCAAA ATAATCATTC CCTGCCTAGCACTTCTCTTC 2700 TGGCCATGGA ACTAAGTACA GTGGCACTGT TTGAGGACCA GTGTTCCCGGGGTTCCTGTG 2760 TGCCCTTATT TCTCCTGGAC TTTTCATTTA AGCTCCAAGC CCCAAATCTGGGGGGCTAGT 2820 TTAGAAACTC TCCCTCAACC TAGTTTAGAA ACTCTACCCC ATCTTTAATACCTTGAATGT 2880 TTTGAACCCC ACTTTTTACC TTCATGGGTT GCAGAAAAAT CAGAACAGATGTCCCCATCC 2940 ATGCGATTGC CCCACCATCT ACTAATGAAA AATTGTTCTT TTTTTCATCTTTCCCCTGCA 3000 CTTATGTTAC TATTCTCTGC TCCCAGCCTT CATCCTTTTC TAAAAAGGAGCAAATTCTCA 3060 CTCTAGGCTT TATCGTGTTT ACTTTTTCAT TACACTTGAC TTGATTTTCTAGTTTTCTAT 3120 ACAAACACCA ATGGGTTCCA TCTTTCTGGG CTCCTGATTG CTCAAGCACAGTTTGGCCTG 3180 ATGAAGAGGA TTTCAACTAC ACAATACTAT CATTGTCAGG ACTATGACCTCAGGCACTCT 3240 AAACATATGT TTTGTTTGGT CAGCACAGCG TTTCAAAAAG TGAAGCCACTTTATAAATAT 3300 TTGGAGATTT TGCAGGAAAA TCTGGATCCC CAGGTAAGGA TAGCAGATGGTTTTCAGTTA 3360 TCTCCAGTCC ACGTTCACAA AATGTGAAGG TGTGGAGACA CTTACAAAGCTGCCTCACTT 3420 CTCACTGTAA ACATTAGCTC TTTCCACTGC CTACCTGGAC CCCAGTCTAGGAATTAAATC 3480 TGCACCTAAC CAAGGTCCCT TGTAAGAAAT GTCCATTCAA GCAGTCATTCTCTGGGTATA 3540 TAATATGATT TTGACTACCT TATCTGGTGT TAAGATTTGA AGTTGGCCTTTTATTGGACT 3600 AAAGGGGAAC TCCTTTAAGG GTCTCAGTTA GCCCAAGTTT CTTTTGCTTATATGTTAATA 3660 GTTTTACCCT CTGCATTGGA GAGAGGAGTG CTTTACTCCA AGAAGCTTTCCTCATGGTTA 3720 CCGTTCTCTC CATCATGCCA GCCTTCTCAA CCTTTGCAGA AATTACTAGAGAGGATTTGA 3780 ATGTGGGACA CAAAGGTCCC ATTTGCAGTT AGAAAATTTG TGTCCACAAGGACAAGAACA 3840 AAGTATGAGC TTTAAAACTC CATAGGAAAC TTGTTAATCA ACAAAGAAGTGTTAATGCTG 3900 CAAGTAATCT CTTTTTTAAA ACTTTTTGAA GCTACTTATT TTCAGCCAAATAGGAATATT 3960 AGAGAGGGAC TGGTAGTGAG AATATCAGCT CTGTTTGGAT GGTGGAAGGTCTCATTTTAT 4020 TGAGATTTTT AAGATACATG CAAAGGTTTG GAAATAGAAC CTCTAGGCACCCTCCTCAGT 4080 GTGGGTGGGC TGAGAGTTAA AGACAGTGTG GCTGCAGTAG CATAGAGGCGCCTAGAAATT 4140 CCACTTGCAC CGTAGGGCAT GCTGATACCA TCCCAATAGC TGTTGCCCATTGACCTCTAG 4200 TGGTGAGTTT CTAGAATACT GGTCCATTCA TGAGATATTC AAGATTCAAGAGTATTCTCA 4260 CTTCTGGGTT ATCAGCATAA ACTGGAATGT AGTGTCAGAG GATACTGTGGCTTGTTTTGT 4320 TTATGTTTTT TTTTCTTATT CAAGAAAAAA GACCAAGGAA TAACATTCTGTAGTTCCTAA 4380 AAATACTGAC TTTTTTCACT ACTATACATA AAGGGAAAGT TTTATTCTTTTATGGAACAC 4440 TTCAGCTGTA CTCATGTATT AAAATAGGAA TGTGAATGCT ATATACTCTTTTTATATCAA 4500 AAGTCTCAAG CACTTATTTT TATTCTATGC ATTGTTTGTC TTTTACATAAATAAAATGTT 4560 TATTAGATTG AATAAAGCAA AATACTCAGG TGAGCATCCT GCCTCCTGTTCCCATTCCTA 4620 GTAGCTAAA 4629 Seq. ID No. 3: Homo sapiens transforminggrowth factor, beta receptor II (TGFBR2), transcript variant 2, mRNA(sense; written in RNA code) GGAGAGGGAG AAGGCUCUCG GGCGGAGAGA GGUCCUGCCCAGCUGUUGGC GAGGAGUUUC 60 CUGUUUCCCC CGCAGCGCUG AGUUGAAGUU GAGUGAGUCACUCGCGCGCA CGGAGCGACG 120 ACACCCCCGC GCGUGCACCC GCUCGGGACA GGAGCCGGACUCCUGUGCAG CUUCCCUCGG 180 CCGCCGGGGG CCUCCCCGCG CCUCGCCGGC CUCCAGGCCCCCUCCUGGCU GGCGAGCGGG 240 CGCCACAUCU GGCCCGCACA UCUGCGCUGC CGGCCCGGCGCGGGGUCCGG AGAGGGCGCG 300 GCGCGGAGGC GCAGCCAGGG GUCCGGGAAG GCGCCGUCCGCUGCGCUGGG GGCUCGGUCU 360 AUGACGAGCA GCGGGGUCUG CCAUGGGUCG GGGGCUGCUCAGGGGCCUGU GGCCGCUGCA 420 CAUCGUCCUG UGGACGCGUA UCGCCAGCAC GAUCCCACCGCACGUUCAGA AGUCGGUUAA 480 UAACGACAUG AUAGUCACUG ACAACAACGG UGCAGUCAAGUUUCCACAAC UGUGUAAAUU 540 UUGUGAUGUG AGAUUUUCCA CCUGUGACAA CCAGAAAUCCUGCAUGAGCA ACUGCAGCAU 600 CACCUCCAUC UGUGAGAAGC CACAGGAAGU CUGUGUGGCUGUAUGGAGAA AGAAUGACGA 660 GAACAUAACA CUAGAGACAG UUUGCCAUGA CCCCAAGCUCCCCUACCAUG ACUUUAUUCU 720 GGAAGAUGCU GCUUCUCCAA AGUGCAUUAU GAAGGAAAAAAAAAAGCCUG GUGAGACUUU 780 CUUCAUGUGU UCCUGUAGCU CUGAUGAGUG CAAUGACAACAUCAUCUUCU CAGAAGAAUA 840 UAACACCAGC AAUCCUGACU UGUUGCUAGU CAUAUUUCAAGUGACAGGCA UCAGCCUCCU 900 GCCACCACUG GGAGUUGCCA UAUCUGUCAU CAUCAUCUUCUACUGCUACC GCGUUAACCG 960 GCAGCAGAAG CUGAGUUCAA CCUGGGAAAC CGGCAAGACGCGGAAGCUCA UGGAGUUCAG 1020 CGAGCACUGU GCCAUCAUCC UGGAAGAUGA CCGCUCUGACAUCAGCUCCA CGUGUGCCAA 1080 CAACAUCAAC CACAACACAG AGCUGCUGCC CAUUGAGCUGGACACCCUGG UGGGGAAAGG 1140 UCGCUUUGCU GAGGUCUAUA AGGCCAAGCU GAAGCAGAACACUUCAGAGC AGUUUGAGAC 1200 AGUGGCAGUC AAGAUCUUUC CCUAUGAGGA GUAUGCCUCUUGGAAGACAG AGAAGGACAU 1260 CUUCUCAGAC AUCAAUCUGA AGCAUGAGAA CAUACUCCAGUUCCUGACGG CUGAGGAGCG 1320 GAAGACGGAG UUGGGGAAAC AAUACUGGCU GAUCACCGCCUUCCACGCCA AGGGCAACCU 1380 ACAGGAGUAC CUGACGCGGC AUGUCAUCAG CUGGGAGGACCUGCGCAAGC UGGGCAGCUC 1440 CCUCGCCCGG GGGAUUGCUC ACCUCCACAG UGAUCACACUCCAUGUGGGA GGCCCAAGAU 1500 GCCCAUCGUG CACAGGGACC UCAAGAGCUC CAAUAUCCUCGUGAAGAACG ACCUAACCUG 1560 CUGCCUGUGU GACUUUGGGC UUUCCCUGCG UCUGGACCCUACUCUGUCUG UGGAUGACCU 1620 GGCUAACAGU GGGCAGGUGG GAACUGCAAG AUACAUGGCUCCAGAAGUCC UAGAAUCCAG 1680 GAUGAAUUUG GAGAAUGUUG AGUCCUUCAA GCAGACCGAUGUCUACUCCA UGGCUCUGGU 1740 GCUCUGGGAA AUGACAUCUC GCUGUAAUGC AGUGGGAGAAGUAAAAGAUU AUGAGCCUCC 1800 AUUUGGUUCC AAGGUGCGGG AGCACCCCUG UGUCGAAAGCAUGAAGGACA ACGUGUUGAG 1860 AGAUCGAGGG CGACCAGAAA UUCCCAGCUU CUGGCUCAACCACCAGGGCA UCCAGAUGGU 1920 GUGUGAGACG UUGACUGAGU GCUGGGACCA CGACCCAGAGGCCCGUCUCA CAGCCCAGUG 1980 UGUGGCAGAA CGCUUCAGUG AGCUGGAGCA UCUGGACAGGCUCUCGGGGA GGAGCUGCUC 2040 GGAGGAGAAG AUUCCUGAAG ACGGCUCCCU AAACACUACCAAAUAGCUCU UCUGGGGCAG 2100 GCUGGGCCAU GUCCAAAGAG GCUGCCCCUC UCACCAAAGAACAGAGGCAG CAGGAAGCUG 2160 CCCCUGAACU GAUGCUUCCU GGAAAACCAA GGGGGUCACUCCCCUCCCUG UAAGCUGUGG 2220 GGAUAAGCAG AAACAACAGC AGCAGGGAGU GGGUGACAUAGAGCAUUCUA UGCCUUUGAC 2280 AUUGUCAUAG GAUAAGCUGU GUUAGCACUU CCUCAGGAAAUGAGAUUGAU UUUUACAAUA 2340 GCCAAUAACA UUUGCACUUU AUUAAUGCCU GUAUAUAAAUAUGAAUAGCU AUGUUUUAUA 2400 UAUAUAUAUA UAUAUCUAUA UAUGUCUAUA GCUCUAUAUAUAUAGCCAUA CCUUGAAAAG 2460 AGACAAGGAA AAACAUCAAA UAUUCCCAGG AAAUUGGUUUUAUUGGAGAA CUCCAGAACC 2520 AAGCAGAGAA GGAAGGGACC CAUGACAGCA UUAGCAUUUGACAAUCACAC AUGCAGUGGU 2580 UCUCUGACUG UAAAACAGUG AACUUUGCAU GAGGAAAGAGGCUCCAUGUC UCACAGCCAG 2640 CUAUGACCAC AUUGCACUUG CUUUUGCAAA AUAAUCAUUCCCUGCCUAGC ACUUCUCUUC 2700 UGGCCAUGGA ACUAAGUACA GUGGCACUGU UUGAGGACCAGUGUUCCCGG GGUUCCUGUG 2760 UGCCCUUAUU UCUCCUGGAC UUUUCAUUUA AGCUCCAAGCCCCAAAUCUG GGGGGCUAGU 2820 UUAGAAACUC UCCCUCAACC UAGUUUAGAA ACUCUACCCCAUCUUUAAUA CCUUGAAUGU 2880 UUUGAACCCC ACUUUUUACC UUCAUGGGUU GCAGAAAAAUCAGAACAGAU GUCCCCAUCC 2940 AUGCGAUUGC CCCACCAUCU ACUAAUGAAA AAUUGUUCUUUUUUUCAUCU UUCCCCUGCA 3000 CUUAUGUUAC UAUUCUCUGC UCCCAGCCUU CAUCCUUUUCUAAAAAGGAG CAAAUUCUCA 3060 CUCUAGGCUU UAUCGUGUUU ACUUUUUCAU UACACUUGACUUGAUUUUCU AGUUUUCUAU 3120 ACAAACACCA AUGGGUUCCA UCUUUCUGGG CUCCUGAUUGCUCAAGCACA GUUUGGCCUG 3180 AUGAAGAGGA UUUCAACUAC ACAAUACUAU CAUUGUCAGGACUAUGACCU CAGGCACUCU 3240 AAACAUAUGU UUUGUUUGGU CAGCACAGCG UUUCAAAAAGUGAAGCCACU UUAUAAAUAU 3300 UUGGAGAUUU UGCAGGAAAA UCUGGAUCCC CAGGUAAGGAUAGCAGAUGG UUUUCAGUUA 3360 UCUCCAGUCC ACGUUCACAA AAUGUGAAGG UGUGGAGACACUUACAAAGC UGCCUCACUU 3420 CUCACUGUAA ACAUUAGCUC UUUCCACUGC CUACCUGGACCCCAGUCUAG GAAUUAAAUC 3480 UGCACCUAAC CAAGGUCCCU UGUAAGAAAU GUCCAUUCAAGCAGUCAUUC UCUGGGUAUA 3540 UAAUAUGAUU UUGACUACCU UAUCUGGUGU UAAGAUUUGAAGUUGGCCUU UUAUUGGACU 3600 AAAGGGGAAC UCCUUUAAGG GUCUCAGUUA GCCCAAGUUUCUUUUGCUUA UAUGUUAAUA 3660 GUUUUACCCU CUGCAUUGGA GAGAGGAGUG CUUUACUCCAAGAAGCUUUC CUCAUGGUUA 3720 CCGUUCUCUC CAUCAUGCCA GCCUUCUCAA CCUUUGCAGAAAUUACUAGA GAGGAUUUGA 3780 AUGUGGGACA CAAAGGUCCC AUUUGCAGUU AGAAAAUUUGUGUCCACAAG GACAAGAACA 3840 AAGUAUGAGC UUUAAAACUC CAUAGGAAAC UUGUUAAUCAACAAAGAAGU GUUAAUGCUG 3900 CAAGUAAUCU CUUUUUUAAA ACUUUUUGAA GCUACUUAUUUUCAGCCAAA UAGGAAUAUU 3960 AGAGAGGGAC UGGUAGUGAG AAUAUCAGCU CUGUUUGGAUGGUGGAAGGU CUCAUUUUAU 4020 UGAGAUUUUU AAGAUACAUG CAAAGGUUUG GAAAUAGAACCUCUAGGCAC CCUCCUCAGU 4080 GUGGGUGGGC UGAGAGUUAA AGACAGUGUG GCUGCAGUAGCAUAGAGGCG CCUAGAAAUU 4140 CCACUUGCAC CGUAGGGCAU GCUGAUACCA UCCCAAUAGCUGUUGCCCAU UGACCUCUAG 4200 UGGUGAGUUU CUAGAAUACU GGUCCAUUCA UGAGAUAUUCAAGAUUCAAG AGUAUUCUCA 4260 CUUCUGGGUU AUCAGCAUAA ACUGGAAUGU AGUGUCAGAGGAUACUGUGG CUUGUUUUGU 4320 UUAUGUUUUU UUUUCUUAUU CAAGAAAAAA GACCAAGGAAUAACAUUCUG UAGUUCCUAA 4380 AAAUACUGAC UUUUUUCACU ACUAUACAUA AAGGGAAAGUUUUAUUCUUU UAUGGAACAC 4440 UUCAGCUGUA CUCAUGUAUU AAAAUAGGAA UGUGAAUGCUAUAUACUCUU UUUAUAUCAA 4500 AAGUCUCAAG CACUUAUUUU UAUUCUAUGC AUUGUUUGUCUUUUACAUAA AUAAAAUGUU 4560 UAUUAGAUUG AAUAAAGCAA AAUACUCAGG UGAGCAUCCUGCCUCCUGUU CCCAUUCCUA 4620 GUAGCUAAA 4629

1-21. (canceled)
 22. An antisense-oligonucleotide consisting of 10 to 28nucleotides and at least two of the 10 to 28 nucleotides are LNAs,wherein the antisense-oligonucleotide has a gapmer structure with 1 to 5LNA units at the 3′ terminal end and 1 to 5 LNA units at the 5′ terminalend and the antisense-oligonucleotide hybridizes with a region of thegene encoding the TGF-R_(II) or with a region of the mRNA encoding theTGF-R_(II), wherein the region of the gene encoding the TGF-R_(II) orthe region of the mRNA encoding the TGF-R_(II) comprises the sequenceTGGTCCATTC (Seq. ID No. 4), and the antisense-oligonucleotide comprisesa sequence hybridizing with said sequence TGGTCCATTC (Seq. ID No. 4) andsalts and optical isomers of said antisense-oligonucleotide, wherein theantisense-oligonucleotide inhibits the expression of TGF-R_(II).
 23. Theantisense-oligonucleotide according to claim 22, wherein theantisense-oligonucleotide hybridizes selectively only with the sequenceTGGTCCATTC (Seq. ID No. 4) of the region of the gene encoding theTGF-R_(II) or of the region of the mRNA encoding the TGF-R_(II).
 24. Theantisense-oligonucleotide according to claim 22, wherein theantisense-oligonucleotide has a length of 12 to 20 nucleotides and/orwherein the antisense-oligonucleotide has phosphate, phosphorothioateand/or phosphorodithioate as internucleotide linkages.
 25. Theantisense-oligonucleotide according to claim 22, wherein theantisense-oligonucleotide is represented by the following generalformula (S6): (Seq. ID No. 100) 5′-N⁷-AATGGACC-N⁸-3′

wherein N⁷ represents: TGAATCTTGAATATCTCATG- (Seq. ID No. 583),GAATCTTGAATATCTCATG- (Seq. ID No. 584), AATCTTGAATATCTCATG- (Seq. ID No.585), ATCTTGAATATCTCATG- (Seq. ID No. 586), TCTTGAATATCTCATG- (Seq. IDNo. 587), CTTGAATATCTCATG- (Seq. ID No. 588), TTGAATATCTCATG- (Seq. IDNo. 589), TGAATATCTCATG- (Seq. ID No. 590), GAATATCTCATG- (Seq. ID No.591), AATATCTCATG- (Seq. ID No. 592), ATATCTCATG- (Seq. ID No. 593),TATCTCATG-, ATCTCATG-, TCTCATG-, CTCATG-, TCATG-, CATG-, ATG-, TG-, orG-; and N8 is selected from: AGTATTCTAGAAACTCACCA, (Seq. ID No. 594),AGTATTCTAGAAACTCACC, (Seq. ID No. 595), AGTATTCTAGAAACTCAC, (Seq. ID No.596), AGTATTCTAGAAACTCA, (Seq. ID No. 597), AGTATTCTAGAAACTC, (Seq. IDNo. 598), AGTATTCTAGAAACT, (Seq. ID No. 599), AGTATTCTAGAAAC, (Seq. IDNo. 600), AGTATTCTAGAAA, (Seq. ID No. 601), AGTATTCTAGAA, (Seq. ID No.602), AGTATTCTAGA, (Seq. ID No. 603), AGTATTCTAG, (Seq. ID No. 604),AGTATTCTA, AGTATTCT, AGTATTC, AGTATT, AGTAT, AGTA, AGT, AG, or A. 26.The antisense-oligonucleotide according to claim 22, wherein the last 2to 4 nucleotides at the 5′ terminal end are LNA nucleotides and the last2 to 4 nucleotides at the 3′ terminal end are LNA nucleotides andbetween the LNA nucleotides at the 5′ terminal end and the LNAnucleotides at the 3′ terminal end at least 6 consecutive nucleotidesare present which are non-LNA nucleotides or which are DNA nucleotides.27. The antisense-oligonucleotide according to claim 25, wherein thelast 2 to 4 nucleotides at the 5′ terminal end are LNA nucleotides andthe last 2 to 4 nucleotides at the 3′ terminal end are LNA nucleotidesand between the LNA nucleotides at the 5′ terminal end and the LNAnucleotides at the 3′ terminal end at least 6 consecutive nucleotidesare present which are non-LNA nucleotides or which are DNA nucleotides.28. The antisense-oligonucleotide according to claim 22, wherein the LNAnucleotides are linked to each other through a phosphorothioate group ora phosphorodithioate group or wherein all nucleotides are linked to eachother through a phosphate group or a phosphorothioate group or aphosphorodithioate group.
 29. The antisense-oligonucleotide according toclaim 25, wherein the LNA nucleotides are linked to each other through aphosphorothioate group or a phosphorodithioate group or wherein allnucleotides are linked to each other through a phosphate group or aphosphorothioate group or a phosphorodithioate group.
 30. Theantisense-oligonucleotide according to claim 22, wherein the LNAnucleotides are selected from the group consisting of:

wherein IL′ represents —X″—P(═X′)(X⁻)—; X′ represents ═O or ═S; Xrepresents —O⁻, —OH, —OR^(H), —NHR^(H), —N(R^(H))₂, —OCH₂CH₂OR^(H),—OCH₂CH₂SR^(H), —BH₃ ⁻, —R^(H), —SH, —SR^(H), or —S⁻; X″ represents —O—,—NH—, —NR^(H)—, —CH₂—, or —S—; Y is —O—, —NH—, —NR^(H)—, —CH₂— or —S—;R^(C) and R^(H) are independently of each other selected from hydrogenand C₁₋₄-alkyl; and B represents a nucleobase selected from the groupconsisting of: adenine, thymine, guanine, cytosine, uracil,5-methylcytosine, 5-hydroxymethyl cytosine, N⁴-methylcytosine, xanthine,hypoxanthine, 7-deazaxanthine, 2-aminoadenine, 6-methyladenine,6-methylguanine, 6-ethyladenine, 6-ethylguanine, 2-propyladenine,2-propylguanine, 6-carboxyuracil, 5,6-dihydrouracil, 5-propynyl uracil,5-propynyl cytosine, 6-aza uracil, 6-aza cytosine, 6-aza thymine,5-uracil, 4-thiouracil, 8-fluoroadenine, 8-chloroadenine,8-bromoadenine, 8-iodoadenine, 8-aminoadenine, 8-thioladenine,8-thioalkyladenine, 8-hydroxyladenine, 8-fluoroguanine, 8-chloroguanine,8-bromoguanine, 8-iodoguanine, 8-aminoguanine, 8-thiolguanine,8-thioalkylguanine, 8-hydroxylguanine, 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, 5-trifluoromethyluracil, 5-fluorocytosine,5-bromocytosine, 5-chlorocytosine, 5-iodocytosine,5-trifluoromethylcytosine, 7-methylguanine, 7-methyladenine,8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine,7-deaza-8-azaadenine, 3-deazaguanine, 3-deazaadenine, 2-thiouracil,2-thiothymine, and 2-thiocytosine.
 31. The antisense-oligonucleotideaccording to claim 25, wherein the LNA nucleotides are selected from thegroup consisting of:

wherein IL′ represents —X″—P(═X′)(X⁻)—; X′ represents ═O or ═S; Xrepresents —O⁻, —OH, —OR^(H), —NHR^(H), —N(R^(H))₂, —OCH₂CH₂OR^(H),—OCH₂CH₂SR^(H), —BH₃ ⁻, —R^(H), —SH, —SR^(H), or —S⁻; X″ represents —O—,—NH—, —NR^(H)—, —CH₂—, or —S—; Y is —O—, —NH—, —NR^(H)—, —CH₂— or —S—;R^(C) and R^(H) are independently of each other selected from hydrogenand C₁₋₄-alkyl; and B represents a nucleobase selected from the groupconsisting of: adenine, thymine, guanine, cytosine, uracil,5-methylcytosine, 5-hydroxymethyl cytosine, N⁴-methylcytosine, xanthine,hypoxanthine, 7-deazaxanthine, 2-aminoadenine, 6-methyladenine,6-methylguanine, 6-ethyladenine, 6-ethylguanine, 2-propyladenine,2-propylguanine, 6-carboxyuracil, 5,6-dihydrouracil, 5-propynyl uracil,5-propynyl cytosine, 6-aza uracil, 6-aza cytosine, 6-aza thymine,5-uracil, 4-thiouracil, 8-fluoroadenine, 8-chloroadenine,8-bromoadenine, 8-iodoadenine, 8-aminoadenine, 8-thioladenine,8-thioalkyladenine, 8-hydroxyladenine, 8-fluoroguanine, 8-chloroguanine,8-bromoguanine, 8-iodoguanine, 8-aminoguanine, 8-thiolguanine,8-thioalkylguanine, 8-hydroxylguanine, 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, 5-trifluoromethyluracil, 5-fluorocytosine,5-bromocytosine, 5-chlorocytosine, 5-iodocytosine,5-trifluoromethylcytosine, 7-methylguanine, 7-methyladenine,8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine,7-deaza-8-azaadenine, 3-deazaguanine, 3-deazaadenine, 2-thiouracil,2-thiothymine and 2-thiocytosine.
 32. The antisense-oligonucleotideaccording to claim 22 having one of the following gapmer structuresselected from the group consisting of: 3-8-3, 4-8-2, 2-8-4, 3-8-4,4-8-3, 4-8-4, 3-9-3, 4-9-2, 2-9-4, 4-9-3, 3-9-4, 4-9-4, 3-10-3, 2-10-4,4-10-2, 3-10-4, 4-10-3, 4-10-4, 2-11-4, 4-11-2, 3-11-4, and 4-11-3. 33.The antisense-oligonucleotide according to claim 25 having one of thefollowing gapmer structures selected from the group consisting of:3-8-3, 4-8-2, 2-8-4, 3-8-4, 4-8-3, 4-8-4, 3-9-3, 4-9-2, 2-9-4, 4-9-3,3-9-4, 4-9-4, 3-10-3, 2-10-4, 4-10-2, 3-10-4, 4-10-3, 4-10-4, 2-11-4,4-11-2, 3-11-4, and 4-11-3.
 34. The antisense-oligonucleotide accordingto claim 22, wherein the antisense oligonucleotides bind with 100%complementarity to the mRNA encoding TGF-R_(II) and do not bind to anyother region in the human transcriptome.
 35. Theantisense-oligonucleotide according to claim 25, wherein the antisenseoligonucleotides bind with 100% complementarity to the mRNA encodingTGF-R_(II) and do not bind to any other region in the humantranscriptome.
 36. A method for promoting regeneration and functionalreconnection of damaged nerve pathways and/or for treatment andcompensation of age induced decreases in neuronal stem cell renewalcomprising administering to a patient an antisense-oligonucleotideaccording to claim
 22. 37. A method for promoting regeneration andfunctional reconnection of damaged nerve pathways and/or for treatmentand compensation of age induced decreases in neuronal stem cell renewalcomprising administering to a patient an antisense-oligonucleotideaccording to claim
 25. 38. A method for prophylaxis and treatment of adisease selected from the group consisting of neurodegenerativediseases, neuroinflammatory disorders, traumatic or posttraumaticdisorders, neurovascular disorders, hypoxic disorders, postinfectiouscentral nervous system disorders, fibrotic diseases, hyperproliferativediseases, cancer, tumors, presbyakusis and presbyopia comprisingadministering to a patient an antisense-oligonucleotide according toclaim
 22. 39. A method for prophylaxis and treatment of a diseaseselected from the group consisting of neurodegenerative diseases,neuroinflammatory disorders, traumatic or posttraumatic disorders,neurovascular disorders, hypoxic disorders, postinfectious centralnervous system disorders, fibrotic diseases, hyperproliferativediseases, cancer, tumors, presbyakusis and presbyopia comprisingadministering to a patient an antisense-oligonucleotide according toclaim
 25. 40. The method according to claim 38, wherein theneurodegenerative diseases and neuroinflammatory disorders are selectedfrom the group consisting of: Alzheimer's disease, Parkinson's disease,Creutzfeldt Jakob disease, new variant of Creutzfeldt Jakobs disease,Hallervorden Spatz disease, Huntington's disease, multisystem atrophy,dementia, frontotemporal dementia, motor neuron disorders, amyotrophiclateral sclerosis, spinal muscular atrophy, spinocerebellar atrophies,schizophrenia, affective disorders, major depression,meningoencephalitis, bacterial meningoencephalitis, viralmeningoencephalitis, CNS autoimmune disorders, multiple sclerosis, acuteischemic/hypoxic lesions, stroke, CNS and spinal cord trauma, head andspinal trauma, brain traumatic injuries, arteriosclerosis,atherosclerosis, microangiopathic dementia, Binswanger' disease, retinaldegeneration, cochlear degeneration, macular degeneration, cochleardeafness, AIDS related dementia, retinitis pigmentosa, fragile Xassociated tremor/ataxia syndrome, progressive supranuclear palsy,striatonigral degeneration, olivopontocerebellear degeneration, ShyDrager syndrome, age dependant memory deficits, neurodevelopmentaldisorders associated with dementia, Down's Syndrome, synucleinopathies,superoxide dismutase mutations, trinucleotide repeat disorders, trauma,hypoxia, vascular diseases, vascular inflammations, and CNS ageing andwherein the fibrotic diseases are selected from the group consisting of:pulmonary fibrosis, cystic fibrosis, hepatic cirrhosis, endomyocardialfibrosis, old myocardial infarction, atrial fibrosis, mediastinalfibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massivefibrosis, nephrogenic systemic fibrosis, Crohn's Disease, keloid,systemic sclerosis, arthrofibrosis, Peyronie's disease, Dupuytren'scontracture, and residuums after Lupus erythematodes.
 41. The methodaccording to claim 39, wherein the neurodegenerative diseases andneuroinflammatory disorders are selected from the group consisting of:Alzheimer's disease, Parkinson's disease, Creutzfeldt Jakob disease, newvariant of Creutzfeldt Jakobs disease, Hallervorden Spatz disease,Huntington's disease, multisystem atrophy, dementia, frontotemporaldementia, motor neuron disorders, amyotrophic lateral sclerosis, spinalmuscular atrophy, spinocerebellar atrophies, schizophrenia, affectivedisorders, major depression, meningoencephalitis, bacterialmeningoencephalitis, viral meningoencephalitis, CNS autoimmunedisorders, multiple sclerosis, acute ischemic/hypoxic lesions, stroke,CNS and spinal cord trauma, head and spinal trauma, brain traumaticinjuries, arteriosclerosis, atherosclerosis, microangiopathic dementia,Binswanger' disease, retinal degeneration, cochlear degeneration,macular degeneration, cochlear deafness, AIDS related dementia,retinitis pigmentosa, fragile X associated tremor/ataxia syndrome,progressive supranuclear palsy, striatonigral degeneration,olivopontocerebellear degeneration, Shy Drager syndrome, age dependantmemory deficits, neurodevelopmental disorders associated with dementia,Down's Syndrome, synucleinopathies, superoxide dismutase mutations,trinucleotide repeat disorders, trauma, hypoxia, vascular diseases,vascular inflammations, and CNS ageing and wherein the fibrotic diseasesare selected from the group consisting of: pulmonary fibrosis, cysticfibrosis, hepatic cirrhosis, endomyocardial fibrosis, old myocardialinfarction, atrial fibrosis, mediastinal fibrosis, myelofibrosis,retroperitoneal fibrosis, progressive massive fibrosis, nephrogenicsystemic fibrosis, Crohn's Disease, keloid, systemic sclerosis,arthrofibrosis, Peyronie's disease, Dupuytren's contracture, andresiduums after Lupus erythematodes.
 42. A pharmaceutical compositioncomprising at least one antisense-oligonucleotide according to claim 22together with at least one pharmaceutically acceptable carrier,excipient, adjuvant, solvent or diluent.